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Conventional dendritic cells (cDCs) are continuously replenished by bone marrow‐derived precursors called pre‐DCs, which traffic through the blood to peripheral tissues. Pre‐DCs are a heterogeneous population that includes cDC subset‐committed progenitors, namely pre‐cDC1 and pre‐cDC2, which give rise to mature cDC1 and cDC2, respectively. Regulation of pre‐DC subset trafficking is thought to aid the host response to immune challenge. However, the molecular cues regulating pre‐cDC1 versus pre‐cDC2 trafficking toward peripheral sites during homeostasis and disease remain elusive. Here, we report that pre‐cDC1 but not pre‐cDC2 express the T helper type 1‐associated chemokine receptor CXCR3. Moreover, we identify a cell‐intrinsic role for CXCR3 in the trafficking of pre‐cDC1 to melanoma tumors but not to non‐inflamed organs. We also show that tumor cDC1 numbers can be increased pharmacologically by targeting dipeptidyl peptidase‐4 (CD26), a negative regulator of CXCR3 ligands. Our findings demonstrate that pre‐cDC1 trafficking is regulated distinctly from pre‐cDC2, which is relevant for our understanding of the DC lineage in the context of cancer and inflammation. The migration of conventional dendritic cell (cDC) precursors (pre‐DCs) is required for anti‐cancer immunity. The two subsets of cDC, cDC1 and cDC2, derive from committed progenitors called pre‐cDC1 and pre‐cDC2, respectively. In this study, we describe the differential expression of chemokine receptors by pre‐DC subsets and show that pre‐cDC1 but not pre‐cDC2 use CXCR3 to traffic into melanoma tumors in mice. We further show that the number of cDC1 within tumors can be increased through pharmacological inhibition of dipeptidyl‐peptidase 4 (DPP4), which normally inactivates the CXCR3 ligands CXCL9 and CXCL10.

CD4 + T cells can \"help” or \"license” conventional type 1 dendritic cells (cDC1s) to induce CD8 + cytotoxic T lymphocyte (CTL) anticancer responses, as proven in mouse models. We recently identified cDC1s with a transcriptomic imprint of CD4 + T-cell help, specifically in T-cell-infiltrated human cancers, and these cells were associated with a good prognosis and response to PD-1-targeting immunotherapy. Here, we delineate the mechanism of cDC1 licensing by CD4 + T cells in humans. Activated CD4 + T cells produce IFNβ via the STING pathway, which promotes MHC-I antigen (cross-)presentation by cDC1s and thereby improves their ability to induce CTL anticancer responses. In cooperation with CD40 ligand (L), IFNβ also optimizes the costimulatory and other functions of cDC1s required for CTL response induction. IFN-I-producing CD4 + T cells are present in diverse T-cell-infiltrated cancers and likely deliver “help” signals to CTLs locally, according to their transcriptomic profile and colocalization with “helped/licensed” cDCs and tumor-reactive CD8 + T cells. In agreement with this scenario, the presence of IFN-I-producing CD4 + T cells in the TME is associated with overall survival and the response to PD-1 checkpoint blockade in cancer patients.

Background The manipulation of dendritic cells (DCs) for cancer vaccination has not reached its full potential, despite the revolution in cancer immunotherapy. DCs are fundamental for CD8+ T cell activation, which relies on cross-presentation of exogenous antigen on MHC-I and can be fostered by immunogenic cancer cell death. Translational and clinical research has focused on in vitro-generated monocyte-derived DCs, while the vaccination efficacy of natural conventional type 1 DCs (cDC1s), which are associated with improved anti-tumor immunity and specialize on antigen cross-presentation, remains unknown. Methods We isolated primary spleen mouse cDC1s and established a protocol for fast ex vivo activation and antigen-loading with lysates of tumor cells that underwent immunogenic cell death by UV irradiation. Natural tumor antigen-loaded cDC1s were transferred and their potential for induction of endogenous CD8+ and CD4+ T cell responses in vivo, cancer prevention and therapy were assessed in three grafted cancer models. Further, we tested the efficacy of natural cDC1 vaccination in combination and comparison with anti-PD-1 treatment in two “wildtype” tumor models not expressing exogenous antigens. Results Herein, we reveal that primary mouse cDC1s ex vivo loaded with dead tumor cell-derived antigen are activated and induce strong CD8+ T cell responses from the endogenous repertoire upon adoptive transfer in vivo through tumor antigen cross-presentation. Notably, cDC1-based vaccines enhance tumor infiltration by cancer-reactive CD8+ and CD4+ T cells and halt progression of engrafted cancer models, including tumors that are refractory to anti-PD-1 treatment. Moreover, combined tumor antigen-loaded primary cDC1 and anti-PD-1 therapy had strong synergistic effects in a PD-1 checkpoint inhibition susceptible cancer model. Conclusions This preclinical proof-of-principle study is first to support the therapeutic efficacy of cancer immunotherapy with syngeneic dead tumor cell antigen-loaded mouse cDC1s, the equivalents of the human dendritic cell subset that correlates with beneficial prognosis of cancer patients. Our data pave the way for translation of cDC1-based cancer treatments into the clinic when isolation of natural human cDC1s becomes feasible.

Dendritic cells (DCs) play a crucial role in initiating and shaping immune responses. The effects of DCs on adaptive immune responses depend partly on functional specialization of distinct DC subsets, and partly on the activation state of DCs, which is largely dictated by environmental signals. Fully activated immunostimulatory DCs express high levels of costimulatory molecules, produce pro-inflammatory cytokines, and stimulate T cell proliferation, whereas tolerogenic DCs express low levels of costimulatory molecules, produce immunomodulatory cytokines and impair T cell proliferation. Relevant to the increasing use of immune checkpoint blockade in cancer treatment, signals generated from inhibitory checkpoint molecules on DC surface may also contribute to the inhibitory properties of tolerogenic DCs. Yet, our knowledge on the expression of inhibitory molecules on human DC subsets is fragmentary. Therefore, in this study, we investigated the expression of three immune checkpoints on peripheral blood DC subsets, in basal conditions and upon exposure to pro-inflammatory and anti-inflammatory stimuli, by using a flow cytometric panel that allows a direct comparison of the activatory/inhibitory phenotype of DC-lineage and inflammatory DC subsets. We demonstrated that functionally distinct DC subsets are characterized by differential expression of activatory and inhibitory molecules, and that cDC1s in particular are endowed with a unique immune checkpoint repertoire characterized by high TIM-3 expression, scarce PD-L1 expression and lack of ILT2. Notably, this unique cDC1 repertoire was subverted in a group of patients with myelodysplastic syndromes included in the study. Applied to the characterization of DCs in the tumor microenvironment, this panel has the potential to provide valuable information to be used for investigating the role of DC subsets in cancer, guiding DC-targeting treatments, and possibly identifying predictive biomarkers for clinical response to cancer immunotherapy.

This research investigates the regulatory role of the transcription factor PU.1 in type 1 conventional dendritic cells (cDC1) and its therapeutic potential of modulating the nuclear factor kappaB (NF-κB) cells signaling pathway in non-small cell lung cancer (NSCLC). Utilizing single-cell transcriptome sequencing and comprehensive bioinformatics tools, including the CIBERSORT algorithm, we analyzed the immune cell landscape within NSCLC tissues. Our analysis revealed distinct NSCLC subtypes and delineated the developmental trajectories and functional distinctions of cDC1 cells. Key differentially expressed genes (DEGs) and pivotal functional modules within these cells were identified, highlighting PU.1 as a critical mediator underexpressed in NSCLC samples. Functionally, PU.1 demonstrated the induction of the NF-κB pathway, which led to inhibited tumor proliferation and enhanced activation of cDC1, thereby suggesting its role in tumor immune surveillance. In vivo models confirmed the suppressive effect of PU.1 on NSCLC progression, mediated through its influence on cDC1 functionality via the NF-κB pathway. These findings propose PU.1 as a promising target for NSCLC therapeutic strategies, emphasizing the importance of transcriptional regulators in the tumor microenvironment. [Display omitted] •Revealing the critical role of cDC1 in NSCLC progression.•Identifying PU.1 as a potential regulatory gene for NSCLC for the first time.•Demonstrating that PU.1 inhibits tumor cell growth via the NF-κB pathway.•Providing a new therapeutic target for NSCLC.•Conducting in-depth analysis using single-cell transcriptome sequencing.

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.

Cryptococcus neoformans causes severe meningoencephalitis, accounting for an estimated 200,000 deaths each year. Central to mounting an effective defense against these infections is T-cell-mediated immunity, which is orchestrated by dendritic cells (DCs). The knowledge about the role of specific DC subsets in shaping anti-cryptococcal immunity is limited. Here, we demonstrate that Batf3 cDC1s are important drivers of protective Th1 CD4 T-cell responses required for clearance of cryptococcal infection. Deficiency of Batf3 cDC1 in the infected mice leads to significantly reduced Th1 response and exacerbated fungal growth to the point where depleting the remaining CD4 T cells no longer affects fungal burden. Unveiling this pivotal role of cDC1 in antifungal defense is likely to be important for the development of vaccines and therapies against life-threatening fungal pathogens.

Therapeutic vaccines capable of eliciting CD8 T cell responses are a promising approach in cancer, but the magnitude of immune responses to peptide-based vaccine technologies has so far been modest in humans. The cationic liposome adjuvant CAF®09b has recently shown promising results in clinical trials, where it is administered intraperitoneally (i.p.), as preclinical studies demonstrated superior CD8 T cell responses when using this route compared to subcutaneous delivery. Exploring the mechanism of CAF09b in mice we investigated biodistribution of the adjuvant and associated antigen in murine studies. We further examined local innate cell recruitment and CD8 T cell responses in the peritoneal cavity. We observed that i.p. injected CAF09b associated with visceral fatty tissues and created a vaccine depot in the peritoneal cavity. This led to recruitment of BATF3-dependent conventional type I dendritic cells (cDC1) displaying a migratory cDC1 phenotype (CD11c+XCR1+CD103+). Gene ontology analysis further revealed similarities with visceral adipose tissue DCs. CAF09b injection i.p. led to early priming of CD8 T cells localized to the peritoneal cavity and this response was resistant to FTY720 treatment. This study demonstrates that adjuvants can facilitate recruitment of cDC1s to the peritoneal cavity, a feature that may contribute to the effectiveness of i.p. administration on elicitation of CD8 T cell responses. Furthermore, we demonstrate that CAF09b-induced CD8 T cell responses require BATF3-dependent cDC1 cells. Understanding cDC1 and CD8 T cell dynamics via different immunization routes may aid in the design of more effective vaccine strategies. This work was primarily supported by the Danish Research Council (FTP fund no. 9041-00131b). •Adjuvants administered i.p. effectively recruits migratory cDC1 cells to the peritoneal cavity.•Intraperitoneal injection of CAF09b induces bone marrow mobilization of cDC1 progenitors.•CAF09b injection i.p. led to early priming of CD8 T cells localized to the peritoneal cavity.•Priming of CD8 T cell responses by CAF09b requires cDC1 cells.

Atherosclerosis is characterized by lipid accumulation within plaques, leading to foam cell formation and an inflammatory response within the aortic lesions. Lipid disorders have been extensively investigated, however, the cellular and molecular mechanisms that trigger the inflammatory response in atherosclerotic plaques remain far from being fully understood. Xcr1 + cDC1 cells are newly identified antigen-presenting cells in activating immune cells. However, the role of cDC1 cells in atherosclerosis development remains highly controversial. We first confirmed the presence of cDC1 within human atherosclerotic plaques and discovered a significant association between the increasing cDC1 numbers and atherosclerosis progression in mice. Subsequently, we established Xcr1 Cre-Gfp Rosa26 LSL-DTA Apoe –/– mice, a novel and complex genetic model, in which cDC1 was constitutively depleted in vivo during atherosclerosis development. Intriguingly, we observed a notable reduction in atherosclerotic lesions in hyperlipidemic mice, alongside suppressed T cell activation of both CD4 + and CD8 + subsets in the aortic plaques. Notably, aortic macrophages and serum lipid levels were not significantly changed in the cDC1-depleted mice. Single-cell RNA sequencing revealed heterogeneity of Xcr1 + cDC1 cells across the aorta and lymphoid organs under hyperlipidemic conditions. As Xcr1 is the sole receptor for Xcl1, we next explored to target Xcr1 + cDC1 cells via Xcl1 by establishing Xcl1 –/– Apoe –/– mice. Xcl1 –/– Apoe –/– mice exhibited decreased atherosclerotic plaque formation and reduced aortic cDC1 accumulation, indicating that Xcl1 contributes to cDC1-mediated atherosclerotic lesion development. Our results reveal crucial roles of cDC1 in atherosclerosis progression and provide insights into the development of immunotherapies by targeting cDC1 through Xcl1.

Historically platelets are mostly known for their crucial contribution to hemostasis, but there is growing understanding of their role in inflammation and immunity. The immunomodulatory role of platelets entails interaction with pathogens, but also with immune cells including macrophages and dendritic cells (DCs), to activate adaptive immune responses. In our previous work, we have demonstrated that splenic CD169 + macrophages scavenge liposomes and collaborate with conventional type 1 DCs (cDC1) to induce expansion of CD8 + T cells. Here, we show that platelets associate with liposomes and bind to DNGR-1/Clec9a and CD169/Siglec-1 receptors in vitro . In addition, platelets interacted with splenic CD169 + macrophages and cDC1 and further increased liposome internalization by cDC1. Most importantly, platelet depletion prior to liposomal immunization resulted in significantly diminished antigen-specific CD8 + T cell responses, but not germinal center B cell responses. Previously, complement C3 was shown to be essential for platelet-mediated CD8 + T cell activation during bacterial infection. However, after liposomal vaccination CD8 + T cell priming was not dependent on complement C3. While DCs from platelet-deficient mice exhibited unaltered maturation status, they did express lower levels of CCR7. In addition, in the absence of platelets, CCL5 plasma levels were significantly reduced. Overall, our findings demonstrate that platelets engage in a cross-talk with CD169 + macrophages and cDC1 and emphasize the importance of platelets in induction of CD8 + T cell responses in the context of liposomal vaccination.