Migration Of Dendritic Cells Research Articles

Immunophenotypic and Functional Characterization of Eosinophil and Migratory Dendritic Cell Subsets during Filarial Manifestation of Tropical Pulmonary Eosinophilia.

The role of eosinophil and migratory dendritic cell (migDC) subsets during tropical pulmonary eosinophilia (TPE), a potentially fatal complication of lymphatic filariasis, has not been explored. We show that the onset of TPE is characterized by the accumulation of ROS and anaphylatoxins and a rapid influx of morphologically distinct Siglec-Fint resident eosinophils (rEos) and Siglec-Fhi inflammatory eosinophils (iEos) in the lungs, BAL fluid, and blood of TPE mice. While rEos display regulatory behavior, iEos are highly inflammatory cells, as evident in upregulated expression of activation markers CD69 and CD101, anaphylatoxin receptor C5AR1, alarmins s100a8 and s100a9, components of NADPH oxidase, and copious secretion of TNF-α, IFN-γ, IL-6, IL-1β, IL-4, IL-10, IL-12, and TGF-β. Importantly, iEos exhibited heightened ROS generation, higher phagocytic and increased antigen presentation capacity, elevated Ca2+ influx, and increased F-actin polymerization but downregulated negative regulators of the immune response, i.e., Cd300a, Anaxa1, Runx3, Lilrb3, and Serpinb1a, underlining their essential role in promoting lung damage during TPE. Interestingly, TPE mice also showed significant expansion of CD24+CD11b+ migDCs, which showed upregulated expression of maturation and costimulatory markers CD40, CD80, CD83, CD86, and MHCII, increased antigen presentation capacity, and higher migratory potential as evidenced by increased expression of cytokine receptors CCR4, CCR5, CXCR4, and CXCR5. CD24+CD11b+ migDCs also upregulated the expression of immunoregulators PD-L1 and PD-L2 and secreted proinflammatory cytokines, suggesting their significant involvement during TPE. Taken together, we document important morphological, immunophenotypic, and functional characteristics of eosinophil and migDC subsets in the lungs of TPE mice and suggest that they contribute to worsening lung histopathological conditions during TPE.

A machine learning approach to discover migration modes and transition dynamics of heterogeneous dendritic cells.

Dendritic cell (DC) migration is crucial for mounting immune responses. Immature DCs (imDCs) reportedly sense infections, while mature DCs (mDCs) move quickly to lymph nodes to deliver antigens to T cells. However, their highly heterogeneous and complex innate motility remains elusive. Here, we used an unsupervised machine learning (ML) approach to analyze long-term, two-dimensional migration trajectories of Granulocyte-macrophage colony-stimulating factor (GMCSF)-derived bone marrow-derived DCs (BMDCs). We discovered three migratory modes independent of the cell state: slow-diffusive (SD), slow-persistent (SP), and fast-persistent (FP). Remarkably, imDCs more frequently changed their modes, predominantly following a unicyclic SD→FP→SP→SD transition, whereas mDCs showed no transition directionality. We report that DC migration exhibits a history-dependent mode transition and maturation-dependent motility changes are emergent properties of the dynamic switching of the three migratory modes. Our ML-based investigation provides new insights into studying complex cellular migratory behavior.

Manipulation of host immune defenses by effector proteins delivered from multiple secretion systems of Salmonella and its application in vaccine research.

Salmonella is an important zoonotic bacterial species and hazardous for the health of human beings and livestock globally. Depending on the host, Salmonella can cause diseases ranging from gastroenteritis to life-threatening systemic infection. In this review, we discuss the effector proteins used by Salmonella to evade or manipulate four different levels of host immune defenses: commensal flora, intestinal epithelial-mucosal barrier, innate and adaptive immunity. At present, Salmonella has evolved a variety of strategies against host defense mechanisms, among which various effector proteins delivered by the secretory systems play a key role. During its passage through the digestive system, Salmonella has to face the intact intestinal epithelial barrier as well as competition with commensal flora. After invasion of host cells, Salmonella manipulates inflammatory pathways, ubiquitination and autophagy processes with the help of effector proteins. Finally, Salmonella evades the adaptive immune system by interfering the migration of dendritic cells and interacting with T and B lymphocytes. In conclusion, Salmonella can manipulate multiple aspects of host defense to promote its replication in the host.

Abstract 3416: Suppression of AXL inhibits tumor growth through promotion of dendritic cell migration in lung cancer

Abstract AXL is a receptor tyrosine kinase (RTK) that has a various role in tumor progression such as proliferation, metastasis, angiogenesis, drug resistance and immune tolerance. Although targeting AXL exhibits an anti-cancer effects partially depending on the immune system, their molecular mechanisms have not been fully elucidated. To search the roles of AXL in immune tolerance of tumor, we generated AXL knockout (KO) mice. These AXL KO mice showed the reduced tumor growth in syngeneic LLC-1 model compared to wild type mice. To validate the mechanisms underlying the inhibition of AXL KO-derived tumor growth, we examined tumor-infiltrating lymphocytes (TIL). Compared with tumor-bearing wild type mouse, CD45-positive cells were significantly enhanced in tumor-bearing AXL KO mouse. Especially, these results were dominant in CD8-positive cells and dendritic cells (DCs). Tumor-infiltrating DCs showed the reduction of PD-L1 and Tim3 expression. In addition, the inhibition of AXL led to induce the DCs migration. In conclusion, our data demonstrated that targeting AXL may inhibit tumor growth through the activation of DCs including the enhancement of migration and reduced expression of immune check point proteins. Citation Format: Da-Som Kim, Cha Won Lee, Kyungtaek Im, Yun Jung Choi, Dong Ha Kim, Chang-Min Choi, Wonjun Ji, Jae Cheol Lee, Jin Kyung Rho. Suppression of AXL inhibits tumor growth through promotion of dendritic cell migration in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3416.

Gain‑of‑function of IDO in DCs inhibits Tcell immunity by metabolically regulating surface molecules and cytokines.

Both tolerogenicity and immunogenicity of dendritic cells (DCs) are regulated by their intracellular metabolism. As a rate-limiting enzyme of tryptophan (Trp) metabolism, indoleamine 2,3-dioxygenase (IDO) is involved in regulating the functions of numerous cell types, including DCs, a subset of which has a high capacity for producing IDO to control over-activated inflammation. To identify the mechanisms of IDO in DCs, stable DC lines with both gain- and reduction-of-function of IDO were established using a recombinant DNA technique. Although the IDO variation did not affect DC survival and migration, it altered Trp metabolism and other features of DCs analyzed by high-performance liquid chromatography and flow cytometry. On the surface of the DCs, IDO inhibited co-stimulatory CD86 but promoted co-inhibitory programmed cell death ligand 1 expression, and suppressed the antigen uptake, which ultimately led to the compromised ability of DCs to activate T cells. Furthermore, IDO also suppressed IL-12 secretion but enhanced that of IL-10 in DCs, which eventually induced T cells into tolerogenic phenotypes by inhibiting the differentiation of Th1 but promoting that of regulatory T cells. Collectively, the findings of the present study demonstrated that IDO is a key molecule for tolerogenic DC induction by metabolically regulating surface molecule and cytokine expression. This conclusion may lead to the targeted development of therapeutic drugs for autoimmune diseases.

Efficacy of topically applied rapamycin-loaded redox-sensitive nanocarriers in a human skin/T cell co-culture model.

Rapamycin, also known as Sirolimus, is a promising anti-proliferative drug, but its therapeutic use for the topical treatment of inflammatory, hyperproliferative skin disorders is limited by insufficient penetration rates due to its high molecular weight (MW of 914.172g/mol) and high lipophilicity. We have shown that core multi-shell (CMS) nanocarriers sensitive to oxidative environment can improve drug delivery to the skin. In this study, we investigated the mTOR inhibitory activity of these oxidation-sensitive CMS (osCMS) nanocarrier formulations in an inflammatory ex vivo human skin model. In this model, features of inflamed skin were introduced by treating the ex vivo tissue with low-dose serine protease (SP) and lipopolysaccharide (LPS), while phorbol 12-myristate 13-acetate and ionomycin were used to stimulate IL-17A production in the co-cultured SeAx cells. Furthermore, we tried to elucidate the effects of rapamycin on single cell populations isolated from skin (keratinocytes, fibroblast) as well as on SeAx cells. Further, we measured possible effects of the rapamycin formulations on dendritic cell (DC) migration and activation. The inflammatory skin model enabled the assessment of biological readouts at both the tissue and T cell level. All investigated formulations successfully delivered rapamycin across the skin as revealed by reduced IL-17A levels. Nevertheless, only the osCMS formulations reached higher anti-inflammatory effects in the skin compared to the control formulations with a significant downregulation of mTOR activity. These results indicate that osCMS formulations could help to establish rapamycin, or even other drugs with similar physico-chemical properties, in topical anti-inflammatory therapy.

Oral allergy induction through skin exposure to previously tolerated food antigens in murine models

Food allergies (FAs) are caused by a failure of the immune system to regulate oral tolerance (OT). The use of soap containing hydrolyzed wheat overrides acquired OT to wheat through skin exposure. However, in mouse models, the experimental OT is robust, suggesting that acquired OT to allergens prevents the development of FAs. We aimed to analyze the mechanisms and developed a mouse model of FA that overrides acquired OT via skin exposure. Three murine FA models (intraperitoneal [IP], epicutaneous [EC], and intradermal [ID]) were compared to evaluate if allergies to ovalbumin (OVA) that had been previously tolerated orally could be induced. In the ID model, OT was overridden, and allergic reactions of severe anaphylaxis were developed. To analyze this effect in the ID model, we measured the migration of dendritic cells (DCs) into lymph nodes. The induction of OT promoted the migration of CD103+ dermal DCs; moreover, repeated percutaneous doses of OVA for sensitization gradually increased the migration of CD11b+ dermal DCs. The difference in the proportion of regulatory T cells between ID-sensitized groups at the first ID injection disappeared at the tenth injection. Although OT was robust in the IP model, ID sensitization was found to override OT.

A Dendrimer Peptide (KK2DP7) Delivery System with Dual Functions of Lymph Node Targeting and Immune Adjuvants as a General Strategy for Cancer Immunotherapy

The clinical efficacy of personalized cancer vaccines still needs to be improved due to their insufficient immune effect. The development of innovative adjuvants and lymph node-targeted delivery systems is the key to improving the clinical efficacy of personalized vaccines. However, there is still a lack of an adjuvant delivery system that is simple in preparation and capable of mass production and integrates adjuvant and lymph node targeted delivery functions. Here, this work reports that a simple dendrimer polypeptide (KK2DP7) nanoparticle enhances the immune efficacy of an OVA/neoantigen-based vaccine. Due to its multiple functions as a delivery vehicle, immune adjuvant, and facilitator of dendritic cell migration, KK2DP7 efficiently increases the efficiency of antigen uptake and cross-presentation by antigen-presenting cells (APCs) and delivers antigens to lymph nodes via APCs. Strikingly, the antitumor effect of KK2DP7/OVA is superior to that of commonly used adjuvants such as poly(I:C), CpG, and aluminum adjuvant combined with OVA. Furthermore, KK2DP7/OVA combined with anti-PD-1 antibody is able to prevent tumor recurrence in a postoperative recurrent tumor model. Thus, KK2DP7-based cancer vaccines alone or in combination with immune checkpoint blockade therapies to treat tumors or postoperative tumor recurrence are a powerful strategy to enhance antitumor immunity.

CCL5-producing migratory dendritic cells guide CCR5+ monocytes into the draining lymph nodes.

Dendritic cells (DCs) and monocytes capture, transport, and present antigen to cognate T cells in the draining lymph nodes (LNs) in a CCR7-dependent manner. Since only migratory DCs express this chemokine receptor, it is unclear how monocytes reach the LN. In steady-state and following inhalation of several PAMPs, scRNA-seq identified LN mononuclear phagocytes as monocytes, resident, or migratory type 1 and type 2 conventional (c)DCs, despite the downregulation of Xcr1, Clec9a, H2-Ab1, Sirpa, and Clec10a transcripts on migratory cDCs. Migratory cDCs, however, upregulated Ccr7, Ccl17, Ccl22, and Ccl5. Migratory monocytes expressed Ccr5, a high-affinity receptor for Ccl5. Using two tracking methods, we observed that both CD88hiCD26lomonocytes and CD88-CD26hi cDCs captured inhaled antigens in the lung and migrated to LNs. Antigen exposure in mixed-chimeric Ccl5-, Ccr2-, Ccr5-, Ccr7-, and Batf3-deficient mice demonstrated that while antigen-bearing DCs use CCR7 to reach the LN, monocytes use CCR5 to follow CCL5-secreting migratory cDCs into the LN, where they regulate DC-mediated immunity.

Enhanced TLR5-denpendent migration and activation of antigen-loaded airway dendritic cells by flagellin.

Toll-like receptor 5 (TLR5) agonist flagellin is an effective mucosal adjuvant via intranasal (i.n.) administration. Previous studies demonstrated that the mucosal adjuvanticity of flagellin depends on TLR5 signaling of airway epithelial cells. Since dendritic cells (DCs) play a central role in antigen sensitization and the initiation of primary immune responses, we wondered how DCs were modulated by the intranasally administrated flagellin. In this study, a mouse model of i.n. immunization with ovalbumin (OVA), a model antigen, in the presence or absence of flagellin was utilized. We found that nasal administration of flagellin enhanced the coadministered antigen specific antibody responses, and T cell clonal expansion in a TLR5-dependent manner. However, neither the entering of flagellin to nasal lamina propria, nor the uptake of coadministered antigen by nasal resident DCs was associated with TLR5 signaling. In contrary, migration of antigen-loaded DCs from the nasal cavity to the cervical lymph nodes (CLN) and activation of DC in the CLN were both enhanced through TLR5 signaling. Furthermore, for the DCs, flagellin enhanced the expression of CCR7, which was pivotal for DCs in the priming site migrating to draining lymph nodes. Interestingly, the migration, activation and chemokine receptor expression levels of antigen-loaded DC were all significantly higher than that of bystander DCs. In conclusion, intranasally administrated flagellin enhanced TLR5-dependent antigen loaded DCs' migration and activation, but not antigen uptake.

A8 MICROBIAL ACTIVATION OF INTESTINAL DENDRITIC CELLS IS CRITICAL FOR THE ESTABLISHMENT OF NORMAL BEHAVIOR

Abstract Background Accumulating evidence suggests that gut microbiota affects brain development and its function. It is well known that compared with conventional mice (SPF), germ-free (GF) mice display higher exploratory behavior, which normalizes after bacterial colonization. However, little is known about the underlying mechanisms and first critical steps initiating microbiota-gut-brain communication, which lead to establishment of normal behavior. Purpose To investigate the role of immune system in the establishment of normal behavior after bacterial colonization. Method We assessed behavior in GF mice before and after colonization with SPF microbiota, Altered Schaedler Flora (ASF) or the single bacterial strain E. coli JM83, and compared them to SPF mice, using the light-dark preference and tail suspension tests. Levels of brain-derived neurotrophic factor (BDNF) and c-Fos expression were measured by immunofluorescence in the hippocampus and amygdala. Colonic and brain gene expression were assessed using a NanoString technology. The immunodeficient MyD88-/- Ticam1- and SCID mice were used to study the role of the innate and adaptive immune systems. To demonstrate the role of the dendritic cells (DCs), we measured behavior before and after mono-colonization with E. coli JM83 in GF mice treated with cosalane and fingolimod, that inhibit DCs activation and migration, respectively. Brain levels of CD11b, CD11c and CD103 as DCs markers was assessed by immunofluorescence. Result(s) Compared to SPF mice, GF mice showed higher exploratory and less depressive-like behavior. The ex-germ-free mice colonized with ASF microbiota, or mono-colonized with E. coli JM83 showed similar normalization of behavior as those colonized with SPF microbiota. Mono-colonization with E. coli reduced both BDNF and c-fos levels in the hippocampus and amygdala. While colonization of GF SCID mice induced same change in behavior as in wild-type mice, GF MyD88-/-Ticam1-/- mice did not alter their behavior. Mono-colonization affected multiple genes in the colon and the brain, associated with innate immunity and neural plasticity. Treatment with both cosalane and fingolimod prevented behavioral changes after colonization, which was paralleled by absence of CD11b+CD103+CD11c+ cells in the brain, otherwise found in high numbers in control mono-colonized mice and absent in germ-free mice. Conclusion(s) The innate immune system, through activation and migration of intestinal dendritic cells into the brain, initiates the neuro-immune signaling within the gut-brain axis and leads to normalization of behavior after bacterial colonization. Our findings may impact several psychiatric conditions, in which altered innate immune signaling has been implicated. Please acknowledge all funding agencies by checking the applicable boxes below CIHR, Other Please indicate your source of funding; Balsam Family Foundation Disclosure of Interest None Declared

Insights into dendritic cell maturation during infection with application of advanced imaging techniques.

Dendritic cells (DCs) are crucial for the initiation and regulation of adaptive immune responses. When encountering immune stimulus such as bacterial and viral infection, parasite invasion and dead cell debris, DCs capture antigens, mature, acquire immunostimulatory activity and transmit the immune information to naïve T cells. Then activated cytotoxic CD8+ T cells directly kill the infected cells, while CD4+ T helper cells release cytokines to aid the activity of other immune cells, and help B cells produce antibodies. Thus, detailed insights into the DC maturation process are necessary for us to understand the working principle of immune system, and develop new medical treatments for infection, cancer and autoimmune disease. This review summarizes the DC maturation process, including environment sensing and antigen sampling by resting DCs, antigen processing and presentation on the cell surface, DC migration, DC-T cell interaction and T cell activation. Application of advanced imaging modalities allows visualization of subcellular and molecular processes in a super-high resolution. The spatiotemporal tracking of DCs position and migration reveals dynamics of DC behavior during infection, shedding novel lights on DC biology.

Met-Signaling Controls Dendritic Cell Migration in Skin by Regulating Podosome Formation and Function

Signaling through the HGF receptor/Met in skin-resident Langerhans cells (LCs) and dermal dendritic cells (DCs) is essential for their emigration toward draining lymph nodes upon inflammation-induced activation. In this study, we addressed the role of Met signaling in distinct steps of LC/dermal DC emigration from the skin by employing a conditionally Met-deficient mouse model (Metflox/flox). We found that Met deficiency severely impaired podosome formation in DCs and concomitantly decreased the proteolytic degradation of gelatin. Accordingly, Met-deficient LCs failed to efficiently cross the extracellular matrix-rich basement membrane between the epidermis and the dermis. We further observed that HGF-dependent Met activation reduced the adhesion of bone marrow-derived LCs to various extracellular matrix factors and enhanced the motility of DCs in three-dimensional collagen matrices, which was not the case for Met-deficient LCs/DCs. We found no impact of Met signaling on the integrin-independent amoeboid migration of DCs in response to the CCR7 ligand CCL19. Collectively, our data show that the Met-signaling pathway regulates the migratory properties of DC in HGF-dependent and HGF-independent manners.

Immature and mature bone marrow-derived dendritic cells exhibit distinct intracellular mechanical properties

Dendritic cells (DCs) patrol the organism at an immature stage to detect the presence of pathogens. Once activated, these mature DCs reach the lymph nodes to activate antigen-specific T lymphocytes and thus initiate an adaptative immune response to control the pathogen. The migration of both immature and mature DCs is a key process for their optimal function. DC migration requires transit through narrow constrictions that is allowed by their high local and global deformation capabilities. In addition to cytoplasmic changes, the nucleus mechanical properties also have a major impact for cellular migration and motility. Yet, nucleus intracellular mobility of dendritic cells or its variation upon maturation have not been investigated. Our study defines the biophysical phenotypic variations of dendritic cells upon maturation using interferometric deformability cytometry. This method characterizes different cellular mechanical properties, such as elongation and nucleus offset, by assessing the refractive index spatial distribution of shear-induced deformed cells. By using these parameters, our data suggest that in vitro bone marrow derived dendritic cell (BMDC) maturation induces cell stiffening and reduces nucleus mobility, allowing to distinguish immature and mature dendritic cells. Overall, our method provides insights on intracellular mechanical properties of two dendritic cell states.

Network analysis of large-scale ImmGen and Tabula Muris datasets highlights metabolic diversity of tissue mononuclear phagocytes.

The diversity of mononuclear phagocyte (MNP) subpopulations across tissues is one of the key physiological characteristics of the immune system. Here, we focus on understanding the metabolic variability of MNPs through metabolic network analysis applied to three large-scale transcriptional datasets: we introduce (1) an ImmGen MNP open-source dataset of 337 samples across 26 tissues; (2) a myeloid subset of ImmGen Phase I dataset (202 MNP samples); and (3) a myeloid mouse single-cell RNA sequencing (scRNA-seq) dataset (51,364 cells) assembled based on Tabula Muris Senis. To analyze such large-scale datasets, we develop a network-based computational approach, genes and metabolites (GAM) clustering, for unbiased identification of the key metabolic subnetworks based on transcriptional profiles. We define 9 metabolic subnetworks that encapsulate the metabolic differences within MNP from 38 different tissues. Obtained modules reveal that cholesterol synthesis appears particularly active within the migratory dendritic cells, while glutathione synthesis is essential for cysteinyl leukotriene production by peritoneal and lung macrophages.

A novel defined TLR3 agonist as an effective vaccine adjuvant.

Synthetic double-stranded RNA analogs recognized by Toll-like receptor 3 (TLR3) are an attractive adjuvant candidate for vaccines, especially against intracellular pathogens or tumors, because of their ability to enhance T cell and antibody responses. Although poly(I:C) is a representative dsRNA with potent adjuvanticity, its clinical application has been limited due to heterogeneous molecular size, inconsistent activity, poor stability, and toxicity. To overcome these limitations, we developed a novel dsRNA-based TLR3 agonist named NexaVant (NVT) by using PCR-coupled bidirectional in vitro transcription. Agarose gel electrophoresis and reverse phase-HPLC analysis demonstrated that NVT is a single 275-kDa homogeneous molecule. NVT appears to be stable since its appearance, concentration, and molecular size were unaffected under 6 months of accelerated storage conditions. Moreover, preclinical evaluation of toxicity under good laboratory practices showed that NVT is a safe substance without any signs of serious toxicity. NVT stimulated TLR3 and increased the expression of viral nucleic acid sensors TLR3, MDA-5, and RIG-1. When intramuscularly injected into C57BL/6 mice, ovalbumin (OVA) plus NVT highly increased the migration of dendritic cells (DCs), macrophages, and neutrophils into inguinal lymph node (iLN) compared with OVA alone. In addition, NVT substantially induced the phenotypic markers of DC maturation and activation including MHC-II, CD40, CD80, and CD86 together with IFN-β production. Furthermore, NVT exhibited an appropriate adjuvanticity because it elevated OVA-specific IgG, in particular, higher levels of IgG2c (Th1-type) but lower IgG1 (Th2-type). Concomitantly, NVT increased the levels of Th1-type T cells such as IFN-γ+CD4+ and IFN-γ+CD8+ cells in response to OVA stimulation. Collectively, we suggest that NVT with appropriate safety and effectiveness is a novel and promising adjuvant for vaccines, especially those requiring T cell mediated immunity such as viral and cancer vaccines.

A Bidirectional Single-Cell Migration and Retrieval Chip for Quantitative Study of Dendritic Cell Migration.

Dendritic cell (DC) migration is a fundamental step during execution of its adaptive immunity functions. Studying DC migration characteristics is critical for development of DC-dependent allergy treatments, vaccines, and cancer immunotherapies. Here, a microfluidics-based single-cell migration platform is described that enables high-throughput and precise bidirectional cell migration assays. It also allows selective retrieval of cell subpopulations that have different migratory potentials. Using this microfluidic platform, DC migration is investigated in response to different chemoattractants and inhibitors, quantitatively describe DC migration patterns and retrieve DC subpopulations of different migratory potentials for differential gene expression analysis. This platform opens an avenue for precise characterization of cell migration and potential discovery of therapeutic modulators.

Single-cell transcriptomics reveals striking heterogeneity and functional organization of dendritic and monocytic cells in the bovine mesenteric lymph node.

Dendritic and monocytic cells co-operate to initiate and shape adaptive immune responses in secondary lymphoid tissue. The complexity of this system is poorly understood, also because of the high phenotypic and functional plasticity of monocytic cells. We have sequenced mononuclear phagocytes in mesenteric lymph nodes (LN) of three adult cows at the single-cell level, revealing ten dendritic-cell (DC) clusters and seven monocyte/macrophage clusters with clearly distinct transcriptomic profiles. Among DC, we defined LN-resident subsets and their progenitors, as well as subsets of highly activated migratory DC differing in transcript levels for T-cell attracting chemokines. Our analyses also revealed a potential differentiation path for cDC2, resulting in a cluster of inflammatory cDC2 with close transcriptional similarity to putative DC3 and monocyte-derived DC. Monocytes and macrophages displayed sub-clustering mainly driven by pro- or anti-inflammatory expression signatures, including a small cluster of cycling, presumably self-renewing, macrophages. With this transcriptomic snapshot of LN-derived mononuclear phagocytes, we reveal functional properties and differentiation trajectories in a "command center of immunity", and identify elements that are conserved across species.

In Vivo Tracking of Dendritic Cell Migration.

Dendritic cells (DCs) in peripheral tissue serve as a sentinel to invasion and maintain tolerance. They ingest and carry antigens to the draining lymph nodes and present antigens to antigen-specific T cells to initiate acquired immune responses. Thus, understanding DC migration from peripheral tissues and function is critical for understanding DCs' roles in immune homeostasis. Here, we introduced the KikGR in vivo photolabeling system, an ideal tool for monitoring precise cellular movements and related functions in vivo under physiological conditions and during various immune responses that occur in pathologic condition. Using a mouse line expressing photoconvertible fluorescent protein KikGR, we can label DCs in peripheral tissues by changing the color of KikGR from green to red after exposure to violet light and accurately track DC migration from each peripheral tissue to its respective draining lymph nodes.

Double Staining with Fluorescent Tracers to Determine Myeloid Cell Migration of Leishmania-infected Cells from Mouse Skin to Lymphatic Tissues by Flow Cytometry.

Immune cell trafficking in steady-state conditions and inflammatory cell recruitment into injured tissues is crucial for the surveillance of the immune system and the maintenance of body homeostasis. Tracking the cell journey from the infection site in the skin to lymphoid tissues has been challenging, and is typically determined using fluorescent cell tracers, antibodies, or photoconvertible models. Here, we describe the detailed method to track Leishmania-infected myeloid cells migrating from the skin to lymphatic tissues by multiparametric flow cytometry. These methods involve labeling of infective Leishmania donovani parasites with fluorescent cell tracers and phenotyping of myeloid cells with fluorescent antibodies, to determine the infection status of migratory myeloid cells. We also describe the detailed protocol to trace donor monocytes transferred intradermally into recipient mice in Leishmania donovani infection. These protocols can be adapted to study skin-lymphoid tissue migration of dendritic cells, inflammatory monocytes, neutrophils, and other phagocytic myeloid cells in response to vaccine antigens and infection. Key features • Cell-tracking of cell-trace-labeled parasites and monocytes from the skin to lymphatic tissues after transference into donor mice. • Identification of migratory cells labeled with fluorescent cell tracers and antibodies by flow cytometry. • Isolation, labeling, and transference of bone marrow monocytes from donor mice into the skin of recipient mice. • Description of a double-staining technique with fluorescent cell tracers to determine cell and parasite dissemination from the skin to lymphoid tissues.