cDC2 Subsets Research Articles

Optimization of the Irf8 +32-kb enhancer disrupts dendritic cell lineage segregation.

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.

Stat5b/Ezh2 axis governs high PD-L1 expressing tolerogenic dendritic cell subset in autoimmune diabetes

Dendritic cells (DCs) are specialized antigen-presenting cells that play an important role in inducing and maintaining immune tolerance. The altered distribution and/or function of DCs contributes to defective tolerance in autoimmune diseases such as type 1 diabetes (T1D). In human T1D and in NOD mouse models, DCs share some defects and are often described as less tolerogenic and excessively immunogenic. In the NOD mouse model, the autoimmune response is associated with a defect in the Stat5b signaling pathway. We have reported that expressing a constitutively active form of Stat5b in DCs of transgenic NOD mice (NOD.Stat5b-CA), re-established their tolerogenic function, restored autoimmune tolerance and conferred protection from diabetes. However, the role and molecular mechanisms of Stat5b signaling in regulating splenic conventional DCs tolerogenic signature remained unclear. In this study, we reported that, compared to immunogenic splenic DCs of NOD, splenic DCs of NOD.Stat5b-CA mice exhibited a tolerogenic profile marked by elevated PD-L1 and PD-L2 expression, reduced pro-inflammatory cytokine production, increased frequency of the cDC2 subset and decreased frequency of the cDC1 subset. This tolerogenic profile was associated with increased Ezh2 and IRF4 but decreased IRF8 expression. We also found an upregulation of PD-L1 in the cDC1 subset and high PD-L1 and PD-L2 expression in cDC2 of NOD.Stat5b-CA mice. Mechanistically, we demonstrated that Ezh2 plays an important role in the maintenance of high PD-L1 expression in cDC1 and cDC2 subsets and that Ezh2 inhibition resulted in PD-L1 but not PD-L2 downregulation which was more drastic in the cDC2 subset. Additionally, Ezh2 inhibition severely reduced the cDC2 subset and increased the cDC1 subset and Stat5b-CA.DC pro-inflammatory cytokine production. Together our data suggest that the Stat5b-Ezh2 axis is critical for the maintenance of tolerogenic high PD-L1-expressing cDC2 and autoimmune tolerance in NOD.Stat5b-CA mice.

Distinct ontogenetic lineages dictate cDC2 heterogeneity

Conventional dendritic cells (cDCs) include functionally and phenotypically diverse populations, such as cDC1s and cDC2s. The latter population has been variously subdivided into Notch-dependent cDC2s, KLF4-dependent cDC2s, T-bet+ cDC2As and T-bet− cDC2Bs, but it is unclear how all these subtypes are interrelated and to what degree they represent cell states or cell subsets. All cDCs are derived from bone marrow progenitors called pre-cDCs, which circulate through the blood to colonize peripheral tissues. Here, we identified distinct mouse pre-cDC2 subsets biased to give rise to cDC2As or cDC2Bs. We showed that a Siglec-H+ pre-cDC2A population in the bone marrow preferentially gave rise to Siglec-H− CD8α+ pre-cDC2As in tissues, which differentiated into T-bet+ cDC2As. In contrast, a Siglec-H− fraction of pre-cDCs in the bone marrow and periphery mostly generated T-bet− cDC2Bs, a lineage marked by the expression of LysM. Our results showed that cDC2A versus cDC2B fate specification starts in the bone marrow and suggest that cDC2 subsets are ontogenetically determined lineages, rather than cell states imposed by the peripheral tissue environment.

In situ lipid-loading activates peripheral dendritic cell subsets characterized by cellular ROS accumulation but compromises their capacity to prime naïve T cells

Background and aimsDendritic cells (DCs), professional antigen-presenting cells, play an important role in pathologies by controlling adaptive immune responses. However, their adaptation to and functionality in hypercholesterolemia, a driving factor in disease onset and progression of atherosclerosis remains to be established. MethodsIn this study, we addressed the immediate impact of high fat diet-induced hypercholesterolemia in low-density lipoprotein receptor deficient (Ldlr−/−) mice on separate DC subsets, their compartmentalization and functionality. ResultsWhile hypercholesterolemia induced a significant rise in bone marrow myeloid and dendritic cell progenitor (MDP) frequency and proliferation rate after high fat diet feeding, it did not affect DC subset numbers in lymphoid tissue. Hypercholesterolemia led to almost immediate and persistent augmentation in granularity of conventional DCs (cDCs), in particular cDC2, reflecting progressive lipid accumulation by these subsets. Plasmacytoid DCs were only marginally and transiently affected. Lipid loading increased co-stimulatory molecule expression and ROS accumulation by cDC2. Despite this hyperactivation, lipid-laden cDC2 displayed a profoundly reduced capacity to stimulate naïve CD4+ T cells. ConclusionOur data provide evidence that in hypercholesterolemic conditions, peripheral cDC2 subsets engulf lipids in situ, leading to a more activated status characterized by cellular ROS accumulation while, paradoxically, compromising their T cell priming ability. These findings will have repercussions not only for lipid driven cardiometabolic disorders like atherosclerosis, but also for adaptive immune responses to pathogens and/or endogenous (neo) antigens under conditions of hyperlipidemia.

Complement C1q essential for aeroallergen sensitization via CSF1R+ conventional dendritic cells type 2

Dendritic cells (DCs) are heterogeneous, comprising multiple subsets with unique functional specifications. Our previous work has demonstrated that the specific conventional type 2 DC subset, CSF1R+cDC2s, plays a critical role in sensing aeroallergens. It remains to be understood how CSF1R+cDC2s recognize inhaled allergens. We sought to elucidate the transcriptomic programs and receptor-ligand interactions essential for function of this subset in allergen sensitization. We applied single-cell RNA sequencing to mouse lung DCs. Conventional DC-selective knockout mouse models were employed, and mice were subjected to inhaled allergen sensitization with multiple readouts of asthma pathology. Under the clinical arm of this work, human lung transcriptomic data were integrated with mouse data, and bronchoalveolar lavage (BAL) specimens were collected from subjects undergoing allergen provocation, with samples assayed for C1q. We found that C1q is selectively enriched in lung CSF1R+cDC2s, but not in other lung cDC2 or cDC1 subsets. Depletion of C1q in conventional DCs significantly attenuates allergen sensing and features of asthma. Additionally, we found that C1q binds directly to human dust mite allergen, and the C1q receptor CD91 (LRP1) is required for lung CSF1R+cDC2s to recognize the C1q-allergen complex and induce allergic lung inflammation. Lastly, C1q is enriched in human BAL samples following subsegmental allergen challenge, and human RNA sequencing data demonstrate close homology between lung IGSF21+DCs and mouse CSF1R+cDC2s. C1q is secreted from the CSF1R+cDC2 subset among conventional DCs. Our data indicate that the C1q-LRP1 axis represents a candidate for translational therapeutics in the prevention and suppression of allergic lung inflammation.

Saponin-based adjuvants enhance antigen cross-presentation in human CD11c+ CD1c+ CD5− CD163+ conventional type 2 dendritic cells

BackgroundAdjuvants are key for effective vaccination against cancer and chronic infectious diseases. Saponin-based adjuvants (SBAs) are unique among adjuvants in their ability to induce robust cell-mediated immune responses in addition...

Charactering the immune signature of antigen presenting cells in the immune response to factor VIII in a murine model of hemophilia A using single-cell RNA sequencing

Abstract Deficiency of the procoagulant protein factor VIII (FVIII) is the defining characteristic for individuals with the inherited X-linked bleeding disorder hemophilia A. These individuals require intravenous infusions of FVIII to treat bleeding events, however ~30% develop neutralizing alloantibodies (i.e., inhibitors) against exogenous FVIII. Transcriptome analysis of FVIII-stimulated peripheral blood mononuclear cells in patients with hemophilia A and active inhibitors showed increased expression of innate immune modulators and proinflammatory pathways. Murine models of hemophilia A (FVIII KO) similarly develop anti-FVIII antibodies with FVIII exposure. Although the antibody response to FVIII is CD4-T cell dependent, the key antigen presenting cells (APCs) that mediate inhibitor formation is unclear. Bone marrow chimeras of transgenic mice with depleted dendritic cell (DC) subsets in FVIII KO suggest that type 2 classical DCs (cDC2), but not cDC1 or plasmacytoid DCs, mediate FVIII immunity. We then evaluated the evolution of splenic APCs and their immune cell signatures in FVIII KO mice injected with 0, 1, 3, or 5 doses of FVIII using flow cytometry and single-cell RNA sequencing (scRNAseq). FVIII KO injected with 5 doses of FVIII had similar frequencies of pDC, cDC1, and cDC2 subsets compared to mice injected with normal saline as a control. However, preliminary scRNAseq analysis demonstrated increased expression of phagocytic and interferon-related genes among DC and macrophage clusters, particularly CD8a −CD4 +Itgax −DCs. Further scRNAseq analyses of APC subclusters and differentially expressed genes will be assessed. Overall, these findings suggest that cDCs, specifically cDC2, are critical in FVIII immunity. Supported by NIH K99HL150595 and Hemophilia of Georgia Clinical Scientist Development Grant.

Classical DC2 subsets and monocyte-derived DC: Delineating the developmental and functional relationship.

To specifically tailor immune responses to a given pathogenic threat, dendritic cells (DC) are highly heterogeneous and comprise many specialized subtypes, including conventional DC (cDC) and monocyte-derived DC (MoDC), each with distinct developmental and functional characteristics. However, the functional relationship between cDC and MoDC is not fully understood, as the overlapping phenotypes of certain type 2 cDC (cDC2) subsets and MoDC do not allow satisfactory distinction of these cells in the tissue, particularly during inflammation. However, precise cDC2 and MoDC classification is required for studies addressing how these diverse cell types control immune responses and is therefore currently one of the major interests in the field of cDC research. This review will revise murine cDC2 and MoDC biology in the steady state and under inflammatory conditions and discusses the commonalities and differences between ESAMlo cDC2, inflammatory cDC2, and MoDC and their relative contribution to the initiation, propagation, and regulation of immune responses.

Epicutaneous allergen immunotherapy induces a profound and selective modulation in skin dendritic-cell subsets

Epicutaneous immunotherapy (EPIT) protocols have recently been developed to restore tolerance in patients with food allergy. The mechanisms by which EPIT protocols promote desensitization rely on a profound immune deviation of pathogenic T- and B-cell responses. To date, little is known about the contribution of skin dendritic cells (skDCs) to T-cell remodeling and EPIT efficacy. We capitalized on a preclinical model of food allergy to ovalbumin (OVA) to characterize the phenotype and functions of OVA⁺ skDCs throughout the course of EPIT. Our results showed that both Langerhans cells and dermal conventional cDC1 and cDC2 subsets retained their ability to capture OVA in the skin and to migrate toward the skin-draining lymph nodes during EPIT. However, their activation/maturation status was significantly impaired, as evidenced by the gradual and selective reduction of CD86, CD40, and OVA protein expression in respective subsets. Phenotypic changes during EPIT were also characterized by a progressive diversification of single-cell gene signatures within each DC subset. Interestingly, we observed that OVA⁺ Langerhans cells progressively lost their capacity to prime CD4⁺ T_EFF cells, but gained regulatory T-cell stimulatory properties. In contrast, cDC1 were inefficient in priming CD4⁺ T_EFF cells or in reactivating T_MEM cells invitro, whereas cDC2 retained moderate stimulatory properties, and progressively biased type 2 immunity toward type 1 and type 17 responses. Our results therefore emphasize that the acquisition of distinct phenotypic and functional specializations by skDCs during EPIT is at the cornerstone of the desensitization process.

Type 2 cytokines in the thymus activate Sirpα+ dendritic cells to promote clonal deletion.

The thymus contains a diversity of dendritic cells (DCs) that exist in defined locations and have different antigen-processing and -presenting features. This suggests that they play nonredundant roles in mediating thymocyte selection. In an effort to eliminate SIRPα+ classic DC2 subsets, we discovered that a substantial proportion expresses the surface lectin, CD301b, in the thymus. These cells resemble the CD301b+ type 2 immune response promoting DCs that are present in the skin-draining lymph nodes. Transcriptional and phenotypic comparison to other DC subsets in the thymus revealed that thymic CD301b+ cDCs represent an activated state that exhibits enhanced antigen processing and presentation. Furthermore, a CD301b+ cDC2 subset demonstrated a type 2 cytokine signature and required steady-state interleukin-4 receptor signaling. Selective ablation of CD301b+ cDC2 subsets impaired clonal deletion without affecting regulatory T cells (Treg cells). The T cell receptor α repertoire sequencing confirmed that a cDC2 subset promotes deletion of conventional T cells with minimal effect on Treg cell selection. Together, these findings suggest that cytokine-induced activation of DCs in the thymus substantially enforces central tolerance.

Cellular and molecular mediators of thymic DC homeostasis and activation

Abstract Central tolerance in the thymus ensures that autoreactive thymocytes undergo negative selection or diversion to the regulatory T cell (Treg) lineage. Thymic antigen presenting cells (APCs) such as medullary thymic epithelial cells (mTECs) and dendritic cells (DCs) present myriad self-peptides to thymocytes to induce central tolerance. We and others have previously shown that thymic cDCs can be subdivided into activated CCR7+MHC-IIhi and resting CCR7− MHC-IIlo cDC1 and cDC2 subsets with distinct functional properties, suggesting the thymic DC compartment may be more heterogeneous than previously described. Here, we performed single-cell RNA sequencing (scRNAseq) of hematopoietic APCs isolated from adult mouse thymi to comprehensively identify thymic DC subsets with distinct gene expression profiles. Our data reveal 11 transcriptionally distinct cDC subsets. We identify 6 cDC1 subsets, 3 cDC2 subsets, 2 activated CCR7+ “cDC3” subsets distinguished by expression of cDC1 and cDC2 markers, and 4 plasmacytoid DC clusters. Through functional assays, we find that the XCR1+ cDC3s are the most efficient at presenting mTEC-derived peptides on MHC-I and MHC-II, while 2 distinct cDC2 subsets are the most efficient at generating Tregs in vitro. Additional scRNA-seq and flow cytometry analyses of thymic DCs from mice in which thymocyte maturation was blocked at different developmental stages reveal candidate cellular and molecular crosstalk signals that regulate homeostasis and/or activation of pDC and cDC subsets. Our findings reveal hitherto unknown functional heterogeneity within the thymic DC compartment and identify potential interactions that support DC homeostasis and activation which in turn could impact central tolerance. Supported by NIH P01 AI139449-02

An IL-2 mutein promotes Foxp3+ Treg-mediated suppression of dendritic cell activation in response to inflammatory stimuli

Abstract Foxp3+ regulatory T (Treg) cells are essential for maintaining immune tolerance and the development of strategies that boost Tregs is a promising approach for the treatment of autoimmune disorders. We previously developed a murine Fc-fused IL-2 mutein (Fc.Mut24) with decreased affinity for IL-2Rβ resulting in increased CD25 dependency and potent Treg selectivity. High-dose Fc.Mut24 administration prevents autoimmunity in the non-obese diabetic (NOD) mouse; however, the mechanisms by which Fc.Mut24 affects Treg suppressive function remain unclear. In this study, we have performed a phenotypic analysis of Treg and classical dendritic cells (DC) from IL-2 mutein-treated mice. We have found that Fc.Mut24 treatment increases CTLA-4 recycling/expression on Treg with a concurrent decrease in PD-1 expression specifically on effector regulatory T (eTreg) cells. Both CD8+ cDC1 and DCIR2+ cDC2 subsets from Fc.Mut24-treated mice have reduced expression of the co-stimulatory molecules CD80/CD86 following lipopolysaccharide (LPS) injection. Furthermore, RNA-sequencing reveals that Tregs regulate the transcriptional response of DCs to LPS and may preferentially suppress cDC1 activation in vivo. These results suggest that a major mechanism of action by IL-2 mutein may be promoting CTLA-4-mediated transendocytosis of CD80/CD86 while simultaneously reducing PD-1: PD-L1 co-inhibitory signaling between eTreg and DCs. Our findings address unanswered questions about the role of IL-2 in modulating Treg suppressive function and have important implications for the use of Treg-selective IL-2 therapeutics in the clinic. B.L.J. is supported by a postdoctoral fellowship from the Washington Research Foundation.

Clonal lineage tracing reveals shared origin of conventional and plasmacytoid dendritic cells

Developmental origins of dendritic cells (DCs) including conventional DCs (cDCs, comprising cDC1 and cDC2 subsets) and plasmacytoid DCs (pDCs) remain unclear. We studied DC development in unmanipulated adult mice using inducible lineage tracing combined with clonal DNA "barcoding" and single-cell transcriptome and phenotype analysis (CITE-seq). Inducible tracing of Cx3cr1+ hematopoietic progenitors in the bone marrow showed that they simultaneously produce all DC subsets including pDCs, cDC1s, and cDC2s. Clonal tracing of hematopoietic stem cells (HSCs) and of Cx3cr1+ progenitors revealed clone sharing between cDC1s and pDCs, but not between the two cDC subsets or between pDCs and B cells. Accordingly, CITE-seq analyses of differentiating HSCs and Cx3cr1+ progenitors identified progressive stages of pDC development including Cx3cr1+ Ly-6D+ pro-pDCs that were distinct from lymphoid progenitors. These results reveal the shared origin of pDCs and cDCs and suggest a revised scheme of DC development whereby pDCs share clonal relationship with cDC1s.

Methionine- and Choline-Deficient Diet Identifies an Essential Role for DNA Methylation in Plasmacytoid Dendritic Cell Biology.

Diet plays an important role in lifestyle disorders associated with the disturbed immune system. During the study of methionine- and choline-deficient diet-induced nonalcoholic fatty liver disease, we observed a specific decrease in the plasmacytoid dendritic cell (pDC) fraction from murine spleens. While delineating the role for individual components, we identified that l-methionine supplementation correlates with representation of the pDC fraction. S-adenosylmethionine (SAM) is a key methyl donor, and we demonstrate that supplementation of methionine-deficient medium with SAM but not homocysteine reverses the defect in pDC development. l-Methionine has been implicated in maintenance of methylation status in the cell. Based on our observed effect of SAM and zebularine on DC subset development, we sought to clarify the role of DNA methylation in pDC biology. Whole-genome bisulfite sequencing analysis from the splenic DC subsets identified that pDCs display differentially hypermethylated regions in comparison with classical DC (cDC) subsets, whereas cDC1 and cDC2 exhibited comparable methylated regions, serving as a control in our study. We validated differentially methylated regions in the sorted pDC, CD8α+ cDC1, and CD4+ cDC2 subsets from spleens as well as FL-BMDC cultures. Upon analysis of genes linked with differentially methylated regions, we identified that differential DNA methylation is associated with the MAPK pathway such that its inhibition guides DC development toward the pDC subtype. Overall, our study identifies an important role for methionine in pDC biology.

JAK1 signaling in dendritic cells promotes peripheral tolerance in autoimmunity through PD-L1-mediated regulatory Tcell induction.

Dendritic cells (DCs) induce peripheral Tcell tolerance, but cell-intrinsic signaling cascades governing their stable tolerogenesis remain poorly defined. Janus Kinase 1 (JAK1) transduces cytokine-receptor signaling, and JAK inhibitors (Jakinibs), including JAK1-specific filgotinib, break inflammatory cycles in autoimmunity. Here, we report in heterogeneous DC populations of multiple secondary lymphoid organs that JAK1 promotes peripheral Tcell tolerance during experimental autoimmune encephalomyelitis (EAE). Mice harboring DC-specific JAK1 deletion exhibit elevated peripheral CD4+ Tcell expansion, less regulatory Tcells (Tregs), and worse EAE outcomes, whereas adoptive DC transfer ameliorates EAE pathogenesis by inducing peripheral Tregs, programmed cell death ligand 1 (PD-L1) dependently. This tolerogenic program is substantially reduced upon the transfer of JAK1-deficient DCs. DC-intrinsic IFN-γ-JAK1-STAT1 signaling induces PD-L1, which is required for DCs to convert CD4+ Tcells into Tregs invitro and attenuated upon JAK1 deficiency and filgotinib treatment. Thus, DC-intrinsic JAK1 promotes peripheral tolerance, suggesting potential unwarranted DC-mediated effects of Jakinibs in autoimmune diseases.

Early life exposure to house dust mite allergen prevents experimental allergic asthma requiring mitochondrial H2O2.

Immune tolerance to allergens in early-life decreases the risk for asthma in later life. Here we show establishment of stable airway tolerance to the allergen, house dust mite (HDM), by exposing newborn mice repeatedly to a low dose of the allergen. Lung dendritic cells (DCs) from tolerized mice induced a low Th2 response in vitro mirroring impact of tolerance in vivo. In line with our previous finding of increased mitochondrial H2O2 production from lung DCs of mice tolerized to ovalbumin, depletion of mitochondrial H2O2 in MCAT mice abrogated HDM-induced airway tolerance (Tol) with elevated Th2 effector response, airway eosinophilia and increased airway hyperreactivity. WT Tol mice displayed a decrease in total, cDC1 and cDC2 subsets in the lung as compared to that in naïve mice. In contrast, the lungs of MCAT Tol mice showed 3-fold higher numbers of cDCs including those of the subsets as compared to that in WT mice. Our study demonstrates an important role of mitochondrial H2O2 in constraining lung DC numbers towards establishment of early-life airway tolerance to allergens.

The transcription factor C/EBPβ orchestrates dendritic cell maturation and functionality under homeostatic and malignant conditions

Dendritic cell (DC) maturation is a prerequisite for the induction of adaptive immune responses against pathogens and cancer. Transcription factor (TF) networks control differential aspects of early DC progenitor versus late-stage DC cell fate decisions. Here, we identified the TF C/EBPβ as a key regulator for DC maturation and immunogenic functionality under homeostatic and lymphoma-transformed conditions. Upon cell-specific deletion of C/EBPβ in CD11c+MHCIIhi DCs, gene expression profiles of splenic C/EBPβ-/- DCs showed a down-regulation of E2F cell cycle target genes and associated proliferation signaling pathways, whereas maturation signatures were enriched. Total splenic DC cell numbers were modestly increased but differentiation into cDC1 and cDC2 subsets were unaltered. The splenic CD11c+MHCIIhiCD64+ DC compartment was also increased, suggesting that C/EBPβ deficiency favors the expansion of monocytic-derived DCs. Expression of C/EBPβ could be mimicked in LAP/LAP* isoform knockin DCs, whereas the short isoform LIP supported a differentiation program similar to deletion of the full-length TF. In accordance with E2F1 being a negative regulator of DC maturation, C/EBPβ-/- bone marrow-derived DCs matured much faster enabling them to activate and polarize T cells stronger. In contrast to a homeostatic condition, lymphoma-exposed DCs exhibited an up-regulation of the E2F transcriptional pathways and an impaired maturation. Pharmacological blockade of C/EBPβ/mTOR signaling in human DCs abrogated their protumorigenic function in primary B cell lymphoma cocultures. Thus, C/EBPβ plays a unique role in DC maturation and immunostimulatory functionality and emerges as a key factor of the tumor microenvironment that promotes lymphomagenesis.

Migration of murine intestinal dendritic cell subsets upon intrinsic and extrinsic TLR3 stimulation.

Initiation of adaptive immunity to particulate antigens in lymph nodes largely depends on their presentation by migratory dendritic cells (DCs). DC subsets differ in their capacity to induce specific types of immunity, allowing subset-specific DC-targeting to influence vaccination and therapy outcomes. Faithful drug design, however, requires exact understanding of subset-specific versus global activation mechanisms. cDC1, the subset of DCs that excel in supporting immunity toward viruses, intracellular bacteria, and tumors, express uniquely high levels of the pattern recognition receptor TLR3. Using various murine genetic models, we show here that both, the cDC1 and cDC2 subsets of cDCs are activated and migrate equally well in response to TLR3 stimulation in a cell extrinsic and TNF-α dependent manner, but that cDC1 show a unique requirement for type I interferon signaling. Our findings reveal common and differing pathways regulating DC subset migration, offering important insights for the design of DC-based vaccination and therapy approaches.

Tissue-Specific Factors Differentially Regulate the Expression of Antigen-Processing Enzymes During Dendritic Cell Ontogeny.

Dendritic cells (DCs) form a collection of antigen-presenting cells (APCs) that are distributed throughout the body. Conventional DCs (cDCs), which include the cDC1 and cDC2 subsets, and plasmacytoid DCs (pDCs) constitute the two major ontogenically distinct DC populations. The pDCs complete their differentiation in the bone marrow (BM), whereas the cDC subsets derive from pre-committed BM precursors, the pre-cDC, that seed lymphoid and non-lymphoid tissues where they further differentiate into mature cDC1 and cDC2. Within different tissues, cDCs express distinct phenotype and function. Notably, cDCs in the thymus are exquisitely efficient at processing and presenting antigens in the class II pathway, whereas in the spleen they do so only upon maturation induced by danger signals. To appraise this functional heterogeneity, we examined the regulation of the expression of distinct antigen-processing enzymes during DC ontogeny. We analyzed the expression of cathepsin S (CTSS), cathepsin L (CTSL), and thymus-specific serine protease (TSSP), three major antigen-processing enzymes regulating class II presentation in cDC, by DC BM precursors and immature and mature cDCs from the spleen and thymus. We found that pre-cDCs in the BM express relatively high levels of these different proteases. Then, their expression is modulated in a tissue-specific and subset-specific manner with immature and mature thymic cDCs expressing overall higher levels than immature splenic cDCs. On the other hand, the TSSP expression level is selectively down-regulated in spleen pDCs, whereas CTSS and CTSL are both increased in thymic and splenic pDCs. Hence, tissue-specific factors program the expression levels of these different proteases during DC differentiation, thus conferring tissue-specific function to the different DC subsets.

Expression of Siglec-1, -3, -5 and -10 in porcine cDC1 and cDC2 subsets from blood, spleen and lymph nodes and functional capabilities of these cells

Dendritic cells are professional antigen-presenting cells that play a critical role in the development of immune responses. DCs express a variety of Siglecs on their surface, which play a regulatory role modulating their activation through interaction with sialylated structures expressed by cells or pathogens. Here, we characterized the phenotype of porcine conventional dendritic cells subsets from blood, spleen and lymph nodes, emphasizing the analysis of the expression of Siglecs. Siglec-1 was detected in type 1 cDC and, at lower levels, in type 2 cDC in the spleen, being low to negative in blood and lymph node cDC. Siglec-3 and Siglec-5 were expressed in cDC1 at lower levels than in cDC2. Porcine cDCs did not express Siglec-10. cDC2 showed a higher capacity to phagocytose microspheres and to process DQ™-OVA than cDC1, but none of these functions was affected by engagement of Siglec-3 and -5 with antibodies on blood cDC.