Guidelines for preparation and flow cytometry analysis of human nonlymphoid tissue DC.

This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state-of-the-art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy, and functional characterization of mouse and human dendritic cells (DC) from lymphoid organs, and various nonlymphoid tissues. Within this article, detailed protocols are presented that allow for the generation of single-cell suspensions from human nonlymphoid tissues including lung, skin, gingiva, intestine as well as from tumors and tumor-draining lymph nodes with a subsequent analysis of dendritic cells by flow cytometry. Further, prepared single-cell suspensions can be subjected to other applications including cellular enrichment procedures, RNA sequencing, functional assays, etc.

Identification and characterization of murine glycoprotein 2-expressing intestinal dendritic cells.

The intestinal lamina propria (LP) contains distinct subsets of classical dendritic cells (cDC), each playing key non-redundant roles in intestinal immune homeostasis. Here, we show that glycoprotein 2 (GP2), a GPI-anchored protein and receptor for bacterial type-I fimbriae, is selectively expressed by CD103CD11b cDC in the murine small intestine (SI). GP2 expression was induced on CD103CD11b cDC within the SI-LP and was regulated by IRF4, TGFβR1- and retinoic acid signalling.

The small and large intestine contain related mesenchymal subsets that derive from embryonic Gli1 precursors.

The intestinal lamina propria contains a diverse network of fibroblasts that provide key support functions to cells within their local environment. Despite this, our understanding of the diversity, location and ontogeny of fibroblasts within and along the length of the intestine remains incomplete. Here we show that the small and large intestinal lamina propria contain similar fibroblast subsets that locate in specific anatomical niches.

Intragranuloma Accumulation and Inflammatory Differentiation of Neutrophils Underlie Mycobacterial ESX-1-Dependent Immunopathology.

The conserved ESX-1 type VII secretion system is a major virulence determinant of pathogenic mycobacteria, including Mycobacterium tuberculosis and Mycobacterium marinum. ESX-1 is known to interact with infected macrophages, but its potential roles in regulating other host cells and immunopathology have remained largely unexplored. Using a murine M.

cDC1-derived IL-27 regulates small intestinal CD4+ T cell homeostasis in mice.

The small intestinal lamina propria contains large numbers of IFNγ-producing T helper (Th1) cells that play important roles in intestinal homeostasis and host defense, but the mechanisms underlying their development remain poorly understood. Here, we demonstrate that Th1 cells accumulate in the SI-LP after weaning and are maintained there long term. While both Th17 and Th1 cell accumulation in the SI-LP was microbiota dependent, Th1 cell accumulation uniquely required IL-27 and MHCII expression by cDC1.

A self-sustaining layer of early-life-origin B cells drives steady-state IgA responses in the adult gut.

The adult immune system consists of cells that emerged at various times during ontogeny. We aimed to define the relationship between developmental origin and composition of the adult B cell pool during unperturbed hematopoiesis. Lineage tracing stratified murine adult B cells based on the timing of output, revealing that a substantial portion originated within a restricted neonatal window.

IRF8 deficiency induces the transcriptional, functional, and epigenetic reprogramming of cDC1 into the cDC2 lineage.

Conventional dendritic cells (cDCs) consist of two major functionally and phenotypically distinct subsets, cDC1 and cDC2, whose development is dependent on distinct sets of transcription factors. Interferon regulatory factor 8 (IRF8) is required at multiple stages of cDC1 development, but its role in committed cDC1 remains unclear. Here, we used Xcr1-cre to delete Irf8 in committed cDC1 and demonstrate that Irf8 is required for maintaining the identity of cDC1.

ADP-ribosylating adjuvant reveals plasticity in cDC1 cells that drive mucosal Th17 cell development and protection against influenza virus infection.

Migratory dendritic cells expressing CD103 are the targets for mucosal vaccines. These belong to either of two lineage-restricted subsets, cDC1 or cDC2 cells, which have been linked to priming of functionally distinct CD4 T cells. However, recent studies have identified plasticity in cDC2 cells with overlapping functions with cDC1 cells, while the converse has not been reported.

The porcine large intestine contains developmentally distinct submucosal lymphoid clusters and mucosal isolated lymphoid follicles.

Gut-associated lymphoid tissues (GALT) serve as key priming sites for intestinal adaptive immune responses. Most of our understanding of GALT function and development arises from studies in mice. However, the diversity, structure and cellular composition of GALT differs markedly between mammalian species and the developmental window in which distinct GALT structures develop in large mammals remains poorly understood.

Bifidobacterium species associated with breastfeeding produce aromatic lactic acids in the infant gut.

Breastfeeding profoundly shapes the infant gut microbiota, which is critical for early life immune development, and the gut microbiota can impact host physiology in various ways, such as through the production of metabolites. However, few breastmilk-dependent microbial metabolites mediating host-microbiota interactions are currently known. Here, we demonstrate that breastmilk-promoted Bifidobacterium species convert aromatic amino acids (tryptophan, phenylalanine and tyrosine) into their respective aromatic lactic acids (indolelactic acid, phenyllactic acid and 4-hydroxyphenyllactic acid) via a previously unrecognized aromatic lactate dehydrogenase (ALDH).

Intestinal cDC1 drive cross-tolerance to epithelial-derived antigen via induction of FoxP3CD8 T.

Although CD8 T cell tolerance to tissue-specific antigen (TSA) is essential for host homeostasis, the mechanisms underlying peripheral cross-tolerance and whether they may differ between tissue sites remain to be fully elucidated. Here, we demonstrate that peripheral cross-tolerance to intestinal epithelial cell (IEC)-derived antigen involves the generation and suppressive function of FoxP3CD8 T cells. FoxP3CD8 T generation was dependent on intestinal cDC1, whose absence led to a break of tolerance and epithelial destruction.

Human gut-associated lymphoid tissues (GALT); diversity, structure, and function.

Gut-associated lymphoid tissues (GALT) are the key antigen sampling and adaptive immune inductive sites within the intestinal wall. Human GALT includes the multi-follicular Peyer's patches of the ileum, the vermiform appendix, and the numerous isolated lymphoid follicles (ILF) which are distributed along the length of the intestine. Our current understanding of GALT diversity and function derives primarily from studies in mice, and the relevance of many of these findings to human GALT remains unclear.

Identification, isolation and analysis of human gut-associated lymphoid tissues.

Gut-associated lymphoid tissues (GALTs) comprise key intestinal immune inductive sites, including the Peyer's patches of the small intestine and different types of isolated lymphoid follicle (ILF) found along the length of the gut. Our understanding of human GALT is limited due to a lack of protocols for their isolation. Here we describe a technique that, uniquely among intestinal cell isolation protocols, allows identification and isolation of all human GALT, as well as GALT-free intestinal lamina propria (LP).

Lysates of Methylococcus capsulatus Bath induce a lean-like microbiota, intestinal FoxP3RORγtIL-17 Tregs and improve metabolism.

Interactions between host and gut microbial communities are modulated by diets and play pivotal roles in immunological homeostasis and health. We show that exchanging the protein source in a high fat, high sugar, westernized diet from casein to whole-cell lysates of the non-commensal bacterium Methylococcus capsulatus Bath is sufficient to reverse western diet-induced changes in the gut microbiota to a state resembling that of lean, low fat diet-fed mice, both under mild thermal stress (T22 °C) and at thermoneutrality (T30 °C). Concomitant with microbiota changes, mice fed the Methylococcus-based western diet exhibit improved glucose regulation, reduced body and liver fat, and diminished hepatic immune infiltration.

Complete Topological Mapping of a Cellular Protein Interactome Reveals Bow-Tie Motifs as Ubiquitous Connectors of Protein Complexes.

The network topology of a protein interactome is shaped by the function of each protein, making it a resource of functional knowledge in tissues and in single cells. Today, this resource is underused, as complete network topology characterization has proved difficult for large protein interactomes. We apply a matrix visualization and decoding approach to a physical protein interactome of a dendritic cell, thereby characterizing its topology with no prior assumptions of structure.

Immune Profiling of Human Gut-Associated Lymphoid Tissue Identifies a Role for Isolated Lymphoid Follicles in Priming of Region-Specific Immunity.

The intestine contains some of the most diverse and complex immune compartments in the body. Here we describe a method for isolating human gut-associated lymphoid tissues (GALTs) that allows unprecedented profiling of the adaptive immune system in submucosal and mucosal isolated lymphoid follicles (SM-ILFs and M-ILFs, respectively) as well as in GALT-free intestinal lamina propria (LP). SM-ILF and M-ILF showed distinct patterns of distribution along the length of the intestine, were linked to the systemic circulation through MAdCAM-1 high endothelial venules and efferent lymphatics, and had immune profiles consistent with immune-inductive sites.

Deletion of IRF4 in Dendritic Cells Leads to Delayed Onset of T Cell-Dependent Colitis.

Classical dendritic cells (cDC) can be classified into two major subsets: Irf8-dependent cDC1 and Irf4-expressing cDC2. Although these subsets play distinct roles in intestinal immune homeostasis, their functions in T cell-driven colitis remain unknown. To assess the role of IRF4 expression in cDC2 in T cell-driven colitis, CD11c-Cre.

Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition).

These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells.

SMAC mimetics promote NIK-dependent inhibition of CD4 T17 cell differentiation.

Second mitochondria-derived activator of caspase (SMAC) mimetics (SMs) are selective antagonists of the inhibitor of apoptosis proteins (IAPs), which activate noncanonical NF-κB signaling and promote tumor cell death. Through gene expression analysis, we found that treatment of CD4 T cells with SMs during T helper 17 (T17) cell differentiation disrupted the balance between two antagonistic transcription factor modules. Moreover, proteomics analysis revealed that SMs altered the abundance of proteins associated with cell cycle, mitochondrial activity, and the balance between canonical and noncanonical NF-κB signaling.

Intestinal CD103CD11b cDC2 Conventional Dendritic Cells Are Required for Primary CD4 T and B Cell Responses to Soluble Flagellin.

Systemic immunization with soluble flagellin (sFliC) from Typhimurium induces mucosal responses, offering potential as an adjuvant platform for vaccines. Moreover, this engagement of mucosal immunity is necessary for optimal systemic immunity, demonstrating an interaction between these two semi-autonomous immune systems. Although TLR5 and CD103CD11b cDC2 contribute to this process, the relationship between these is unclear in the early activation of CD4 T cells and the development of antigen-specific B cell responses.

Retinoic Acid Signaling in Thymic Epithelial Cells Regulates Thymopoiesis.

Despite the essential role of thymic epithelial cells (TEC) in T cell development, the signals regulating TEC differentiation and homeostasis remain incompletely understood. In this study, we show a key in vivo role for the vitamin A metabolite, retinoic acid (RA), in TEC homeostasis. In the absence of RA signaling in TEC, cortical TEC (cTEC) and CD80MHC class II medullary TEC displayed subset-specific alterations in gene expression, which in cTEC included genes involved in epithelial proliferation, development, and differentiation.

CD103CD11b mucosal classical dendritic cells initiate long-term switched antibody responses to flagellin.

Antibody responses induced at mucosal and nonmucosal sites demonstrate a significant level of autonomy. Here, we demonstrate a key role for mucosal interferon regulatory factor-4 (IRF4)-dependent CD103CD11b (DP), classical dendritic cells (cDCs) in the induction of T-dependent immunoglobulin G (IgG) and immunoglobulin A (IgA) responses in the mesenteric lymph node (MLN) following systemic immunization with soluble flagellin (sFliC). In contrast, IRF8-dependent CD103CD11b (SP) are not required for these responses.

Distinct DC subsets regulate adaptive Th1 and 2 responses during Trichuris muris infection.

Low- and high-dose infections with the murine large intestinal nematode Trichuris muris are associated with induction of adaptive Th1 and Th2 responses, respectively, in mesenteric lymph nodes (MLN). Classical dendritic cells (cDC) accumulate in the large intestinal mucosa and MLN upon T. muris infection, yet their role in driving adaptive responses to infection remains largely unknown.

-dependent CD103CD11b dendritic cells and the intestinal microbiome regulate monocyte and macrophage activation and intestinal peristalsis in postoperative ileus.

Objective: Postoperative ileus (POI), the most frequent complication after intestinal surgery, depends on dendritic cells (DCs) and macrophages. Here, we have investigated the mechanism that activates these cells and the contribution of the intestinal microbiota for POI induction.

Design: POI was induced by manipulating the intestine of mice, which selectively lack DCs, monocytes or macrophages.

Different populations of CD11b dendritic cells drive Th2 responses in the small intestine and colon.

T-helper 2 (Th2) cell responses defend against parasites. Although dendritic cells (DCs) are vital for the induction of T-cell responses, the DC subpopulations that induce Th2 cells in the intestine are unidentified. Here we show that intestinal Th2 responses against Trichuris muris worms and Schistosoma mansoni eggs do not develop in mice with IRF-4-deficient DCs (IRF-4 CD11c-cre).