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Immunoregulatory mechanisms established in the lymphoid organs are vital for preventing autoimmunity. However, the presence of similar mechanisms in non-lymphoid tissues remains unclear. Through transcriptomic and lipidomic analyses, we find a negative association between psoriasis and fatty acid metabolism, as well as Th2 signature. Homeostatic expression of liver X receptor (LXR) and peroxisome proliferator-activated receptor gamma (PPARγ) is essential for maintaining fatty acid metabolism and for conferring resistance to psoriasis in mice. Perturbation of signal transducer and activator of transcription 6 (STAT6) diminishes the homeostatic levels of LXR and PPARγ. Furthermore, mice lacking STAT6, interleukin 4 receptor alpha (IL-4Rα), or IL-13, but not IL-4, exhibit increased susceptibility to psoriasis. Under steady state, innate lymphoid cells (ILCs) are the primary producers of IL-13. In human skin, inhibiting tonic type 2 immunity exacerbates psoriasis-like inflammation and IL-17A, while activating LXR or PPARγ inhibits them. Hence, we propose that tonic type 2 immunity, driven by IL-13-producing ILCs, represents a crucial tissue checkpoint that represses autoimmunity and maintains lipid homeostasis in the skin.
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Autoinmunidad , Receptores X del Hígado , PPAR gamma , Piel , Animales , Piel/inmunología , Piel/metabolismo , Piel/patología , Humanos , Receptores X del Hígado/metabolismo , Ratones , PPAR gamma/metabolismo , Psoriasis/inmunología , Psoriasis/patología , Psoriasis/metabolismo , Ratones Endogámicos C57BL , Interleucina-13/metabolismo , Factor de Transcripción STAT6/metabolismo , Inmunidad Innata , Masculino , Femenino , Linfocitos/inmunología , Linfocitos/metabolismoRESUMEN
BACKGROUND: Tumor-targeted therapy causes impressive tumor regression, but the emergence of resistance limits long-term survival benefits in patients. Little information is available on the role of the myeloid cell network, especially dendritic cells (DC) during tumor-targeted therapy. METHODS: Here, we investigated therapy-mediated immunological alterations in the tumor microenvironment (TME) and tumor-draining lymph nodes (LN) in the D4M.3A preclinical melanoma mouse model (harboring the V-Raf murine sarcoma viral oncogene homolog B (BRAF)V600E mutation) by using high-dimensional multicolor flow cytometry in combination with multiplex immunohistochemistry. This was complemented with RNA sequencing and cytokine quantification to characterize the immune status of the tumors. The importance of T cells during tumor-targeted therapy was investigated by depleting CD4+ or CD8+ T cells in tumor-bearing mice. Tumor antigen-specific T-cell responses were characterized by performing in vivo T-cell proliferation assays and the contribution of conventional type 1 DC (cDC1) to T-cell immunity during tumor-targeted therapy was assessed using Batf3-/- mice lacking cDC1. RESULTS: Our findings reveal that BRAF-inhibitor therapy increased tumor immunogenicity, reflected by an upregulation of genes associated with immune activation. The T cell-inflamed TME contained higher numbers of activated cDC1 and cDC2 but also inflammatory CCR2-expressing monocytes. At the same time, tumor-targeted therapy enhanced the frequency of migratory, activated DC subsets in tumor-draining LN. Even more, we identified a cDC2 population expressing the Fc gamma receptor I (FcγRI)/CD64 in tumors and LN that displayed high levels of CD40 and CCR7 indicating involvement in T cell-mediated tumor immunity. The importance of cDC2 is underlined by just a partial loss of therapy response in a cDC1-deficient mouse model. Both CD4+ and CD8+ T cells were essential for therapy response as their respective depletion impaired therapy success. On resistance development, the tumors reverted to an immunologically inert state with a loss of DC and inflammatory monocytes together with the accumulation of regulatory T cells. Moreover, tumor antigen-specific CD8+ T cells were compromised in proliferation and interferon-γ-production. CONCLUSION: Our results give novel insights into the remodeling of the myeloid landscape by tumor-targeted therapy. We demonstrate that the transient immunogenic tumor milieu contains more activated DC. This knowledge has important implications for the development of future combinatorial therapies.
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Melanoma , Humanos , Animales , Ratones , Melanoma/metabolismo , Linfocitos T CD8-positivos , Proteínas Proto-Oncogénicas B-raf/genética , Células Dendríticas , Antígenos de Neoplasias , Microambiente TumoralRESUMEN
The use of polychromatic immunofluorescent staining on whole-mount skin enables cell type characterization and aids in the delineation of the physiological and immunological strategies used by the skin to combat pathogens. Using whole-mount skin for polychromatic immunofluorescent staining removes the need for histological sectioning and enables the visualization of anatomical structures and immune cell types in three dimensions. Here we present a detailed protocol for immunostaining with fluorescence-conjugated primary antibodies in whole-mount skin to reveal structural landmarks and specific immune cell types using confocal laser scanning microscopy (CLSM) (Basic Protocol 1). The optimized staining panel reveals structural features such as blood vessels (CD31 antibody) and the lymphatic network (LYVE-1 antibody), in combination with MHCII antibodies for antigen-presenting cells (APCs), CD64 for macrophages and monocytes, CD103 for dendritic epidermal T cells (DETC), and CD326 for Langerhans cells (LC). Basic Protocol 2 describes image visualization pipelines using open-source software (ImageJ/FIJI), enabling four visualization options (z-projections, orthogonal views, 3D visualization, and animation). Basic Protocol 3 describes a quantitative analysis pipeline using CellProfiler to characterize the spatial relationship between cell types using mathematical indices such as Spatial Distribution Index (SDI), Neighborhood Frequency (NF), and Normalized Median Evenness (NME). These protocols will enable researchers to stain, record, analyze, and interpret data from whole-mount skin using commercially available reagents in a CLSM-equipped laboratory and freely available analysis software. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Immunofluorescent staining and imaging for whole-mount mouse skin Basic Protocol 2: File rendering and visualization using FIJI Basic Protocol 3: Spatial image analysis using CellProfiler.
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Imagenología Tridimensional , Piel , Animales , Ratones , Imagenología Tridimensional/métodos , Piel/diagnóstico por imagen , Coloración y Etiquetado , Colorantes , Microscopía Confocal/métodosRESUMEN
The issue of what level of contribution warrants authorship, determining a fair order of authors and when and whom to acknowledge in publications is often a cause of debate, and in some instances, has also been a focus of conflict at certain institutions. Shared resource laboratories (SRLs) play a fundamental role in supporting publications, and SRL staff scientists can contribute to numerous areas such as experimental design, sample preparation, data acquisition, data analysis and manuscript drafting and review. However, SRL staff scientists are often unfairly omitted from the author list. To avoid SRLs and SRL staff scientist contributions going unnoticed, the authors have formulated a set of guidelines to aid in the conceptualization and recognition of the technical and intellectual contributions of SRLs. As a better understanding of the role SRL staff scientists play in the achievement of the scientific lead's experimental aims will foster a positive feedback loop, where acknowledgements can lead to more support and funding for SRLs and more engaged SRL staff capable of supporting discoveries and technological innovations that underpin major advancements in the field of life sciences.
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Autoria , Laboratorios , Humanos , Proyectos de InvestigaciónRESUMEN
Recent studies propose that T follicular helper (Tfh) cells possess a high degree of functional plasticity in addition to their well-defined roles in mediating interleukin-4-dependent switching of germinal center B cells to the production of immunoglobulin (Ig)G1 and IgE antibodies. In particular Tfh cells have been proposed to be an essential stage in Th2 effector cell development that are able to contribute to innate type 2 responses. We used CD4-cre targeted deletion of BCL6 to identify the contribution Tfh cells make to tissue Th2 effector responses in models of atopic skin disease and lung immunity to parasites. Ablation of Tfh cells did not impair the development or recruitment of Th2 effector subsets to the skin and did not alter the transcriptional expression profile or functional activities of the resulting tissue resident Th2 effector cells. However, the accumulation of Th2 effector cells in lung Th2 responses was partially affected by BCL6 deficiency. These data indicate that the development of Th2 effector cells does not require a BCL6 dependent step, implying Tfh and Th2 effector populations follow separate developmental trajectories and Tfh cells do not contribute to type 2 responses in the skin.
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Linfocitos T CD4-Positivos , Linfocitos T Colaboradores-Inductores , Diferenciación Celular , Centro Germinal , Linfocitos B , Proteínas Proto-Oncogénicas c-bcl-6/genéticaRESUMEN
Skin functions as a barrier protecting the host against physical, thermal, chemical changes as well as microbial insults. The skin is populated by several immune cell types that are crucial to host defense and to maintain self-tolerance as well as equilibrium with beneficial microbiota. Conventional dendritic cells (cDCs) are antigen-presenting cells that patrol the skin and all other nonlymphoid tissues for self or foreign antigens, and then migrate to draining lymph nodes to initiate T-cell responses. This review article describes recent developments on skin cDC specialization, focusing on the role of IL-13, a cytokine essential to allergic immune responses that is also secreted at steady state by type-2 innate lymphoid cells in healthy skin, and is required for dermal cDC differentiation. Furthermore, we contextualize how different therapeutics that block IL-13 signaling and were recently approved for the treatment of atopic dermatitis might affect cDCs in human skin.
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Células Dendríticas , Inmunidad Innata , Interleucina-13 , Humanos , Interleucina-13/metabolismo , Linfocitos , Piel/patologíaRESUMEN
Most spatial transcriptomics technologies are limited by their resolution, with spot sizes larger than that of a single cell. Although joint analysis with single-cell RNA sequencing can alleviate this problem, current methods are limited to assessing discrete cell types, revealing the proportion of cell types inside each spot. To identify continuous variation of the transcriptome within cells of the same type, we developed Deconvolution of Spatial Transcriptomics profiles using Variational Inference (DestVI). Using simulations, we demonstrate that DestVI outperforms existing methods for estimating gene expression for every cell type inside every spot. Applied to a study of infected lymph nodes and of a mouse tumor model, DestVI provides high-resolution, accurate spatial characterization of the cellular organization of these tissues and identifies cell-type-specific changes in gene expression between different tissue regions or between conditions. DestVI is available as part of the open-source software package scvi-tools ( https://scvi-tools.org ).
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Neoplasias , Transcriptoma , Animales , Perfilación de la Expresión Génica/métodos , Ratones , Neoplasias/genética , Análisis de la Célula Individual/métodos , Programas Informáticos , Transcriptoma/genética , Secuenciación del ExomaRESUMEN
In addition to providing partial protection against pediatric tuberculosis, vaccination with bacille Calmette-Guérin (BCG) has been reported to confer nonspecific resistance to unrelated pulmonary pathogens, a phenomenon attributed to the induction of long-lasting alterations within the myeloid cell compartment. Here, we demonstrate that intravenous, but not subcutaneous, inoculation of BCG protects human-ACE2 transgenic mice against lethal challenge with SARS-CoV-2 (SCV2) and results in reduced viral loads in non-transgenic animals infected with an α variant. The observed increase in host resistance was associated with reductions in SCV2-induced tissue pathology, inflammatory cell recruitment, and cytokine production that multivariate analysis revealed as only partially related to diminished viral load. We propose that this protection stems from BCG-induced alterations in the composition and function of the pulmonary cellular compartment that impact the innate response to the virus and ensuing immunopathology. While intravenous BCG vaccination is not a clinically acceptable practice, our findings provide an experimental model for identifying mechanisms by which nonspecific stimulation of the pulmonary immune response promotes host resistance to SCV2 lethality.
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Vacuna BCG/inmunología , COVID-19/inmunología , SARS-CoV-2/inmunología , Administración Intravenosa , Enzima Convertidora de Angiotensina 2/metabolismo , Animales , Quimiocinas/metabolismo , Humanos , Inflamación/patología , Ratones Endogámicos C57BL , Ratones Transgénicos , Carga ViralRESUMEN
The signals driving the adaptation of type 2 dendritic cells (DC2s) to diverse peripheral environments remain mostly undefined. We show that differentiation of CD11blo migratory DC2s-a DC2 population unique to the dermis-required IL-13 signaling dependent on the transcription factors STAT6 and KLF4, whereas DC2s in lung and small intestine were STAT6-independent. Similarly, human DC2s in skin expressed an IL-4 and IL-13 gene signature that was not found in blood, spleen and lung DCs. In mice, IL-13 was secreted homeostatically by dermal innate lymphoid cells and was independent of microbiota, TSLP or IL-33. In the absence of IL-13 signaling, dermal DC2s were stable in number but remained CD11bhi and showed defective activation in response to allergens, with diminished ability to support the development of IL-4+GATA3+ helper T cells (TH), whereas antifungal IL-17+RORγt+ TH cells were increased. Therefore, homeostatic IL-13 fosters a noninflammatory skin environment that supports allergic sensitization.
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Comunicación Celular , Diferenciación Celular , Interleucina-13/metabolismo , Células de Langerhans/metabolismo , Piel/metabolismo , Células Th17/metabolismo , Células Th2/metabolismo , Alérgenos/farmacología , Animales , Antígeno CD11b/genética , Antígeno CD11b/metabolismo , Células Cultivadas , Bases de Datos Genéticas , Humanos , Interleucina-13/genética , Células de Langerhans/efectos de los fármacos , Células de Langerhans/inmunología , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Factor de Transcripción STAT6/genética , Factor de Transcripción STAT6/metabolismo , Transducción de Señal , Piel/citología , Piel/efectos de los fármacos , Piel/inmunología , Células Th17/efectos de los fármacos , Células Th17/inmunología , Células Th2/efectos de los fármacos , Células Th2/inmunología , TranscriptomaRESUMEN
Early events in the host response to SARS-CoV-2 are thought to play a major role in determining disease severity. During pulmonary infection, the virus encounters both myeloid and epithelioid lineage cells that can either support or restrict pathogen replication as well as respond with host protective versus detrimental mediators. In addition to providing partial protection against pediatric tuberculosis, vaccination with bacille Calmette-Guérin (BCG) has been reported to confer non-specific resistance to unrelated pulmonary pathogens, a phenomenon attributed to the induction of long-lasting alterations within the myeloid cell compartment. Here we demonstrate that prior intravenous, but not subcutaneous, administration of BCG protects human-ACE2 transgenic mice against lethal challenge with SARS-CoV-2 and results in reduced viral loads in non-transgenic animals infected with an alpha variant. The observed increase in host resistance was associated with reductions in SARS-CoV-2-induced tissue pathology, inflammatory cell recruitment and cytokine production that multivariate analysis revealed to be only partially related to diminished viral load. We propose that this protection stems from BCG-induced alterations in the composition and function of the pulmonary cellular compartment that impact the innate response to the virus and the ensuing immunopathology.
RESUMEN
Dendritic cells residing in the skin represent a large family of antigen-presenting cells, ranging from long-lived Langerhans cells (LC) in the epidermis to various distinct classical dendritic cell subsets in the dermis. Through genetic fate mapping analysis and single-cell RNA-sequencing, we have identified a novel separate population of LC-independent CD207+CD326+ LClike cells in the dermis that homed at a slow rate to the lymph nodes (LNs). These LClike cells are long-lived and radio-resistant but, unlike LCs, they are gradually replenished by bone marrow-derived precursors under steady state. LClike cells together with cDC1s are the main migratory CD207+CD326+ cell fractions present in the LN and not, as currently assumed, LCs, which are barely detectable, if at all. Cutaneous tolerance to haptens depends on LClike cells, whereas LCs suppress effector CD8+ T-cell functions and inflammation locally in the skin during contact hypersensitivity. These findings bring new insights into the dynamism of cutaneous dendritic cells and their function opening novel avenues in the development of treatments to cure inflammatory skin disorders.
Our immune cells are constantly on guard to defend and protect us against invading pathogens, such as bacteria and viruses. Specialized immune cells, known as antigen-presenting cells, or APCs, have a key role in this process. They engulf invaders, chew them up, and travel to the closest local lymph node to stimulate other immune cells with small fragments of these pathogens. This ramps up the immune response to control infection and disease. APCs are a large and diverse family of immune cells, which includes dendritic cells and macrophages. Some APCs work as mobile surveillance units, travelling around the body to find new threats. Others embed themselves in particular organs and tissues, such as the skin, to provide local, on-the-spot surveillance. Langerhans cells are one of the main types of APC in the skin and are found in the thin outer layer of the epidermis. While it is commonly believed that Langerhans cells can move from the epidermis to the skin-draining lymph nodes, some seemingly contradictory evidence exists to suggest that this may not be the case. Now, Sheng et al. have investigated this issue by tracking APCs, including Langerhans cells, in the skin of mice. A powerful genetic cell labelling technique allowed them to track the movement of immune cells inside a living mouse. Sheng et al. found that majority of 'real' Langerhans cells did not leave the skin. Yet, a second lookalike cell that shared many of the same features of a Langerhans cell was found in the dermal layer of skin, and this cell could travel to local lymph nodes. Both the original and lookalike cells had distinct and separate roles in the skin. This research, which has uncovered a new type of Langerhans-like immune cell in the skin, may be extremely useful for developing new targeted therapies to boost immune responses during infection; or to suppress inappropriate immune activation that can lead to autoimmune diseases, such as psoriasis.
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Movimiento Celular , Células Dendríticas/citología , Dermis/citología , Células de Langerhans , Animales , Linaje de la Célula/genética , Ganglios Linfáticos/citología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Análisis de Secuencia de ARNRESUMEN
Antigen (Ag)-presenting cells (APC) instruct CD4+ helper T (Th) cell responses, but it is unclear whether different APC subsets contribute uniquely in determining Th differentiation in pathogen-specific settings. Here, we use skin-relevant, fluorescently-labeled bacterial, helminth or fungal pathogens to track and characterize the APC populations that drive Th responses in vivo. All pathogens are taken up by a population of IRF4+ dermal migratory dendritic cells (migDC2) that similarly upregulate surface co-stimulatory molecules but express pathogen-specific cytokine and chemokine transcripts. Depletion of migDC2 reduces the amount of Ag in lymph node and the development of IFNγ, IL-4 and IL-17A responses without gain of other cytokine responses. Ag+ monocytes are an essential source of IL-12 for both innate and adaptive IFNγ production, and inhibit follicular Th cell development. Our results thus suggest that Th cell differentiation does not require specialized APC subsets, but is driven by inducible and pathogen-specific transcriptional programs in Ag+ migDC2 and monocytes.
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Factores Reguladores del Interferón/inmunología , Monocitos/inmunología , Piel/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Células Presentadoras de Antígenos/inmunología , Diferenciación Celular , Células Dendríticas/inmunología , Femenino , Factores Reguladores del Interferón/genética , Interferón gamma/genética , Interferón gamma/inmunología , Interleucina-17/genética , Interleucina-17/inmunología , Interleucina-4/genética , Interleucina-4/inmunología , Activación de Linfocitos , Masculino , Ratones , Ratones Endogámicos C57BL , Monocitos/citología , Linfocitos T Colaboradores-Inductores/citologíaRESUMEN
Allergic responses are characterized by the activation of a specific subset of effector CD4+ T cells, the T-helper type 2 (Th2) cells, that respond to harmless environmental antigens causing inflammation and pathology. Th2 cells are also found in the context of parasite infections, where they can mediate parasite clearance and expulsion, and support tissue repair. The process that leads to the activation of Th2 cells in vivo is incompletely understood: while it has become clear that "conventional" dendritic cells are essential antigen-presenting cells for the initiation of Th2 immune responses, the molecules that are expressed by dendritic cells exposed to allergens, and the mediators that are produced as a consequence and signal to naïve CD4+ T cells to promote their development into effector Th2, remain to be defined. Here we summarize recent developments in the identification of the dendritic cell subsets involved in Th2 responses, review potential mechanisms proposed to explain the generation of these immune responses, and discuss the direct and indirect signals that condition dendritic cells to drive the development of Th2 responses during allergen or parasite exposure.
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Células Dendríticas , Hipersensibilidad , Células Th2 , Alérgenos , Células Dendríticas/inmunología , Humanos , Inmunidad , Células Th2/inmunologíaRESUMEN
The phenotypic and functional dichotomy between IRF8+ type 1 and IRF4+ type 2 conventional dendritic cells (cDC1s and cDC2s, respectively) is well accepted; it is unknown how robust this dichotomy is under inflammatory conditions, when additionally monocyte-derived cells (MCs) become competent antigen-presenting cells (APCs). Using single-cell technologies in models of respiratory viral infection, we found that lung cDC2s acquired expression of the Fc receptor CD64 shared with MCs and of IRF8 shared with cDC1s. These inflammatory cDC2s (inf-cDC2s) were superior in inducing CD4+ T helper (Th) cell polarization while simultaneously presenting antigen to CD8+ T cells. When carefully separated from inf-cDC2s, MCs lacked APC function. Inf-cDC2s matured in response to cell-intrinsic Toll-like receptor and type 1 interferon receptor signaling, upregulated an IRF8-dependent maturation module, and acquired antigens via convalescent serum and Fc receptors. Because hybrid inf-cDC2s are easily confused with monocyte-derived cells, their existence could explain why APC functions have been attributed to MCs.
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Plasticidad de la Célula/inmunología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Inmunidad , Macrófagos/inmunología , Macrófagos/metabolismo , Infecciones por Respirovirus/etiología , Presentación de Antígeno , Biomarcadores , Susceptibilidad a Enfermedades , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Inmunofenotipificación , Interferón Tipo I/metabolismo , Monocitos/inmunología , Monocitos/metabolismo , Especificidad de Órganos/inmunología , Receptores Fc/metabolismo , Infecciones por Respirovirus/metabolismo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Factores de Transcripción , Virosis/genética , Virosis/inmunología , Virosis/metabolismo , Virosis/virologíaRESUMEN
The skin is inhabited by several immune cell populations that serve as a first line of defence against pathogen invasion. Amongst these populations are dendritic cells, which play an essential sentinel function by taking up antigen or infectious agents and transporting them to the lymph node for T cell recognition and the priming of immune responses. In this review, we briefly summarise recent advances showing how skin dendritic cells are connected to a network of epithelial and stromal cells, which provide structural support, growth factors, spatial cues, contact with the external environment and the skin microbiome, and favour interactions with other immune cells. We propose that this network creates a unique skin environment that may condition dendritic cell phenotype and function.
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Células Dendríticas , Microbiota , Humanos , Células de Langerhans , Ganglios Linfáticos , PielRESUMEN
Dendritic cells are powerful antigen-presenting cells that are essential for the priming of T cell responses. In addition to providing T-cell-receptor ligands and co-stimulatory molecules for naive T cell activation and expansion, dendritic cells are thought to also provide signals for the differentiation of CD4+ T cells into effector T cell populations. The mechanisms by which dendritic cells are able to adapt and respond to the great variety of infectious stimuli they are confronted with, and prime an appropriate CD4+ T cell response, are only partly understood. It is known that in the steady-state dendritic cells are highly heterogenous both in phenotype and transcriptional profile, and that this variability is dependent on developmental lineage, maturation stage, and the tissue environment in which dendritic cells are located. Exposure to infectious agents interfaces with this pre-existing heterogeneity by providing ligands for pattern-recognition and toll-like receptors that are variably expressed on different dendritic cell subsets, and elicit production of cytokines and chemokines to support innate cell activation and drive T cell differentiation. Here we review current information on dendritic cell biology, their heterogeneity, and the properties of different dendritic cell subsets. We then consider the signals required for the development of different types of Th immune responses, and the cellular and molecular evidence implicating different subsets of dendritic cells in providing such signals. We outline how dendritic cell subsets tailor their response according to the infectious agent, and how such transcriptional plasticity enables them to drive different types of immune responses.
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Presentación de Antígeno/inmunología , Células Dendríticas/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Diferenciación Celular/inmunología , Humanos , Modelos Biológicos , Transcripción GenéticaRESUMEN
Crosstalk between neighboring cells underlies many biological processes, including cell signaling, proliferation and differentiation. Current single-cell genomic technologies profile each cell separately after tissue dissociation, losing information on cell-cell interactions. In the present study, we present an approach for sequencing physically interacting cells (PIC-seq), which combines cell sorting of physically interacting cells (PICs) with single-cell RNA-sequencing. Using computational modeling, PIC-seq systematically maps in situ cellular interactions and characterizes their molecular crosstalk. We apply PIC-seq to interrogate diverse interactions including immune-epithelial PICs in neonatal murine lungs. Focusing on interactions between T cells and dendritic cells (DCs) in vitro and in vivo, we map T cell-DC interaction preferences, and discover regulatory T cells as a major T cell subtype interacting with DCs in mouse draining lymph nodes. Analysis of T cell-DC pairs reveals an interaction-specific program between pathogen-presenting migratory DCs and T cells. PIC-seq provides a direct and broadly applicable technology to characterize intercellular interaction-specific pathways at high resolution.
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Células Dendríticas/citología , Perfilación de la Expresión Génica/métodos , Análisis de la Célula Individual/métodos , Linfocitos T/citología , Algoritmos , Animales , Animales Recién Nacidos , Comunicación Celular , Células Cultivadas , Biología Computacional , Células Dendríticas/química , Femenino , Citometría de Flujo , Pulmón/química , Pulmón/citología , Ratones , Análisis de Secuencia de ARN , Linfocitos T/químicaRESUMEN
Colorectal cancer is a major contributor to death and disease worldwide. The ApcMin mouse is a widely used model of intestinal neoplasia, as it carries a mutation also found in human colorectal cancers. However, the method most commonly used to quantify tumour burden in these mice is manual adenoma counting, which is time consuming and poorly suited to standardization across different laboratories. We describe a method to produce suitable photographs of the small intestine of ApcMin mice, process them with an ImageJ macro, FeatureCounter, which automatically locates image features potentially corresponding to adenomas, and a machine learning pipeline to identify and quantify them. Compared to a manual method, the specificity (or True Negative Rate, TNR) and sensitivity (or True Positive Rate, TPR) of this method in detecting adenomas are similarly high at about 80% and 87%, respectively. Importantly, total adenoma area measures derived from the automatically-called tumours were just as capable of distinguishing high-burden from low-burden mice as those established manually. Overall, our strategy is quicker, helps control experimenter bias, and yields a greater wealth of information about each tumour, thus providing a convenient route to getting consistent and reliable results from a study.