Clonal multi-omics reveals Bcor as a negative regulator of emergency dendritic cell development.

Despite advances in single-cell multi-omics, a single stem or progenitor cell can only be tested once. We developed clonal multi-omics, in which daughters of a clone act as surrogates of the founder, thereby allowing multiple independent assays per clone. With SIS-seq, clonal siblings in parallel "sister" assays are examined either for gene expression by RNA sequencing (RNA-seq) or for fate in culture. We identified, and then validated using CRISPR, genes that controlled fate bias for different dendritic cell (DC) subtypes. This included Bcor as a suppressor of plasmacytoid DC (pDC) and conventional DC type 2 (cDC2) numbers during Flt3 ligand-mediated emergency DC development. We then developed SIS-skew to examine development of wild-type and Bcor-deficient siblings of the same clone in parallel. We found Bcor restricted clonal expansion, especially for cDC2s, and suppressed clonal fate potential, especially for pDCs. Therefore, SIS-seq and SIS-skew can reveal the molecular and cellular mechanisms governing clonal fate.

Guidelines for mouse and human DC generation.

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 non-lymphoid tissues. This article provides protocols with top ticks and pitfalls for preparation and successful generation of mouse and human DC from different cellular sources, such as murine BM and HoxB8 cells, as well as human CD34+ cells from cord blood, BM, and peripheral blood or peripheral blood monocytes. We describe murine cDC1, cDC2, and pDC generation with Flt3L and the generation of BM-derived DC with GM-CSF. Protocols for human DC generation focus on CD34+ cell culture on OP9 cell layers for cDC1, cDC2, cDC3, and pDC subset generation and DC generation from peripheral blood monocytes (MoDC). Additional protocols include enrichment of murine DC subsets, CRISPR/Cas9 editing, and clinical grade human DC generation. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer-reviewed by leading experts and approved by all co-authors, making it an essential resource for basic and clinical DC immunologists.

FLT3 Ligand Is Dispensable for the Final Stage of Type 1 Conventional Dendritic Cell Differentiation.

Conventional dendritic cells (cDCs) are comprised of two major subsets, type 1 cDC (cDC1) and type 2 cDC (cDC2). As each cDC subset differentially influences the nature of immune responses, we sought factors that would allow the manipulation of their relative abundance. Notably, cDC1 are less abundant than cDC2 in both lymphoid and nonlymphoid organs. We demonstrate that this bias is already apparent in bone marrow precommitted precursors. However, comparison of five common inbred strains revealed a disparity in precursor-product relationship, in which mice with fewer precursors to cDC1 had more cDC1. This disparity associated with contrasting variations in CD135 (FLT3) expression on cDC subsets. Hence, we characterized the response to FLT3 ligand during cDC1 and cDC2 lineage differentiation and find that although FLT3 ligand is required throughout cDC2 differentiation, it is surprisingly dispensable during late-stage cDC1 differentiation. Overall, we find that tight regulation of FLT3 ligand levels throughout cDC differentiation dictates the cDC1 to cDC2 ratio in lymphoid organs.

Merocytic Dendritic Cells Compose a Conventional Dendritic Cell Subset with Low Metabolic Activity.

Conventional dendritic cells (cDCs) are arguably the most potent APCs that induce the activation of naive T cells in response to pathogens. In addition, at steady-state, cDCs help maintain immune tolerance. Two subsets of cDCs have been extensively characterized, namely cDC1 and cDC2, each contributing differently to immune responses. Recently, another dendritic cell (DC) subset, termed merocytic DCs (mcDCs), was defined. In contrast to both cDC1 and cDC2, mcDCs reverse T cell anergy, properties that could be exploited to potentiate cancer treatments. Yet, whether mcDCs represent an unconventional DC or a cDC subset remains to be defined. In this article, we further characterize mcDCs and find that they bear true characteristics of cDC subsets. Indeed, as for cDCs, mcDCs express the cDC-restricted transcription factor Zbtb46 and display very potent APC activity. In addition, mcDC population dynamics parallels that of cDC1 and cDC2 in both reconstitution kinetic studies and parabiotic mice. We next investigated their relatedness to cDC1 and cDC2 and demonstrate that mcDCs are not dependent on cDC1-related Irf8 and Batf3 transcription factors, are dependent on Irf4, a cDC2-specific transcription factor, and express a unique transcriptomic signature. Finally, we find that cDC1, cDC2, and mcDCs all present with different metabolic phenotypes, in which mcDCs exhibit the lowest glucose uptake activity and mcDC survival is the least affected by glycolysis inhibition. Defining the properties of mcDCs in mice may help identify a functionally equivalent subset in humans leading to the development of innovative cancer immunotherapies.

CD5 levels reveal distinct basal T-cell receptor signals in T cells from non-obese diabetic mice.

Type 1 diabetes in non-obese diabetic (NOD) mice occurs when autoreactive T cells eliminate insulin producing pancreatic ß cells. While extensively studied in T-cell receptor (TCR) transgenic mice, the contribution of alterations in thymic selection to the polyclonal T-cell pool in NOD mice is not yet resolved. The magnitude of signals downstream of TCR engagement with self-peptide directs the development of a functional T-cell pool, in part by ensuring tolerance to self. TCR interactions with self-peptide are also necessary for T-cell homeostasis in the peripheral lymphoid organs. To identify differences in TCR signal strength that accompany thymic selection and peripheral T-cell maintenance, we compared CD5 levels, a marker of basal TCR signal strength, on immature and mature T cells from autoimmune diabetes-prone NOD and -resistant B6 mice. The data suggest that there is no preferential selection of NOD thymocytes that perceive stronger TCR signals from self-peptide engagement. Instead, NOD mice have an MHC-dependent increase in CD4+ thymocytes and mature T cells that express lower levels of CD5. In contrast, T cell-intrinsic mechanisms lead to higher levels of CD5 on peripheral CD8+ T cells from NOD relative to B6 mice, suggesting that peripheral CD8+ T cells with higher basal TCR signals may have survival advantages in NOD mice. These differences in the T-cell pool in NOD mice may contribute to the development or progression of autoimmune diabetes.

Induced and spontaneous colitis mouse models reveal complex interactions between IL-10 and IL-12/IL-23 pathways.

Crohn's disease (CD) and ulcerative colitis (UC) are the two major forms of inflammatory bowel disease (IBD). These idiopathic and chronic diseases result from inflammation of the gastrointestinal tract and are mainly mediated by the immune system. Genome wide association studies link genes of the IL-12 and IL-23 biology to both CD and UC susceptibility. IL-12 and IL-23 cytokines share a functional subunit, p40, and their respective receptors also share a functional subunit, IL-12Rß1. However, clinical trials targeting p40, and thus inhibiting both IL-12 and IL-23 pathways, provided mitigated effects on IBD, suggesting context dependent effects for each cytokine. In addition to IL-12 and IL-23, genetic deficiencies in IL-10 also result in severe IBD pathology. We generated various mouse models to determine how IL-12 or IL-23 interacts with IL-10 in IBD pathology. Whereas defects in both IL-10 and IL-12R do not impact the severity of the Dextran Sulfate Sodium (DSS)-induced colitis, combined deficiencies in both IL-10 and IL-23R aggravate the disease. In contrast to DSS-induced colitis, defects in IL-12R and IL-23R both protect from the spontaneous colitis observed in IL10-/- mice. Together, these studies exemplify the complexity of genetic and environmental interactions for identifying biological pathways predictive of pathological inflammatory processes.

The Importance of Dendritic Cells in Maintaining Immune Tolerance.

Immune tolerance is necessary to prevent the immune system from reacting against self, and thus to avoid the development of autoimmune diseases. In this review, we discuss key findings that position dendritic cells (DCs) as critical modulators of both thymic and peripheral immune tolerance. Although DCs are important for inducing both immunity and tolerance, increased autoimmunity associated with decreased DCs suggests their nonredundant role in tolerance induction. DC-mediated T cell immune tolerance is an active process that is influenced by genetic variants, environmental signals, as well as the nature of the specific DC subset presenting Ag to T cells. Answering the many open questions with regard to the role of DCs in immune tolerance could lead to the development of novel therapies for the prevention of autoimmune diseases.

BIM determines the number of merocytic dendritic cells, a cell type that breaks immune tolerance.

In contrast to conventional dendritic cells (cDC), when merocytic dendritic cells (mcDC) present antigens derived from apoptotic bodies, T-cell anergy is reversed rather than induced, a process that promotes autoimmunity. Interestingly, mcDC are present in higher proportion in type 1 diabetes-prone NOD mice than in autoimmune-resistant B6 and BALB/c mice, and the Insulin-dependent diabetes (Idd)13 locus is linked to mcDC proportion. Therefore, mcDC are notably associated with susceptibility to autoimmune diabetes. To identify which gene determines the proportion and absolute number of mcDC, we undertook a candidate gene approach by selecting relevant candidates within the Idd13 locus. We find that neither ß2m nor Sirpa appear to influence the proportion of mcDC. Instead, we show that Bim effectively modulates mcDC number in a hematopoietic-intrinsic manner. We also demonstrate that Bim-deficiency does not impact other cDC subsets and appears to play a specific role in determining the proportion and absolute number of mcDC by promoting their survival. Together, these data demonstrate that Bim specifically modulates the number of mcDC. Identifying factors that facilitate apoptosis of mcDC by increasing BIM activity in a cell type-specific manner may help prevent autoimmunity.

Mis-expression of GATA6 re-programs cell fate during early hematopoiesis.

The traditional view of hematopoiesis is that myeloid cells derive from a common myeloid progenitor (CMP), whereas all lymphoid cell populations, including B, T, and natural killer (NK) cells and possibly plasmacytoid dendritic cells (pDCs), arise from a common lymphoid progenitor (CLP). In Max41 transgenic mice, nearly all B cells seem to be diverted into the granulocyte lineage. Here, we show that these mice have an excess of myeloid progenitors, but their CLP compartment is ablated, and they have few pDCs. Nevertheless, T cell and NK cell development proceeds relatively normally. These hematopoietic abnormalities result from aberrant expression of Gata6 due to serendipitous insertion of the transgene enhancer (Eµ) in its proximity. Gata6 mis-expression in Max41 transgenic progenitors promoted the gene-regulatory networks that drive myelopoiesis through increasing expression of key transcription factors, including PU.1 and C/EBPa. Thus, mis-expression of a single key regulator like GATA6 can dramatically re-program multiple aspects of hematopoiesis.

Transcriptional regulation of dendritic cell development and function.

Dendritic cells (DCs) are sentinel immune cells that form a critical bridge linking the innate and adaptive immune systems. Extensive research addressing the cellular origin and heterogeneity of the DC network has revealed the essential role played by the spatiotemporal activity of key transcription factors. In response to environmental signals DC mature but it is only following the sensing of environmental signals that DC can induce an antigen specific T cell response. Thus, whilst the coordinate action of transcription factors governs DC differentiation, sensing of environmental signals by DC is instrumental in shaping their functional properties. In this review, we provide an overview that focuses on recent advances in understanding the transcriptional networks that regulate the development of the reported DC subsets, shedding light on the function of different DC subsets. Specifically, we discuss the emerging knowledge on the heterogeneity of cDC2s, the ontogeny of pDCs, and the newly described DC subset, DC3. Additionally, we examine critical transcription factors such as IRF8, PU.1, and E2-2 and their regulatory mechanisms and downstream targets. We highlight the complex interplay between these transcription factors, which shape the DC transcriptome and influence their function in response to environmental stimuli. The information presented in this review provides essential insights into the regulation of DC development and function, which might have implications for developing novel therapeutic strategies for immune-related diseases.

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.

Death by differentiation: CD4+ T cells kick out suspicious stem cells.

Antigen presentation is typically regarded as the domain of immune cells such as dendritic cells and B cells. Hernandez-Malmierca et al. (2022) upend this notion by observing that hematopoietic stem and progenitor cells process and present antigen via major histocompatibility class II as a means of CD4+ T cell-mediated immune surveillance.

Collagen analogs with phosphorylcholine are inflammation-suppressing scaffolds for corneal regeneration from alkali burns in mini-pigs.

The long-term survival of biomaterial implants is often hampered by surgery-induced inflammation that can lead to graft failure. Considering that most corneas receiving grafts are either pathological or inflamed before implantation, the risk of rejection is heightened. Here, we show that bioengineered, fully synthetic, and robust corneal implants can be manufactured from a collagen analog (collagen-like peptide-polyethylene glycol hybrid, CLP-PEG) and inflammation-suppressing polymeric 2-methacryloyloxyethyl phosphorylcholine (MPC) when stabilized with the triazine-based crosslinker 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride. The resulting CLP-PEG-MPC implants led to reduced corneal swelling, haze, and neovascularization in comparison to CLP-PEG only implants when grafted into a mini-pig cornea alkali burn model of inflammation over 12 months. Implants incorporating MPC allowed for faster nerve regeneration and recovery of corneal sensation. CLP-PEG-MPC implants appear to be at a more advanced stage of regeneration than the CLP-PEG only implants, as evidenced by the presence of higher amounts of cornea-specific type V collagen, and a corresponding decrease in the presence of extracellular vesicles and exosomes in the corneal stroma, in keeping with the amounts present in healthy, unoperated corneas.

BMP9 signaling promotes the normalization of tumor blood vessels.

The presence of an immature tumor vascular network contributes to cancer dissemination and the development of resistance to therapies. Strategies to normalize the tumor vasculature are therefore of significant therapeutic interest for cancer treatments. VEGF inhibitors are used clinically to normalize tumor blood vessels. However, the time frame and dosage of these inhibitors required to achieve normalization is rather narrow, and there is a need to identify additional signaling targets to attain vascular normalization. In addition to VEGF, the endothelial-specific receptor Alk1 plays a critical role in vascular development and promotes vascular remodeling and maturation. Therefore, we sought to evaluate the effects of the Alk1 ligand BMP9 on tumor vascular formation. BMP9 overexpression in Lewis Lung Carcinoma (LLC) tumors significantly delayed tumor growth. Blood vessels in BMP9-overexpressing LLC tumors displayed markers of vascular maturation and were characterized by increased perivascular cell coverage. Tumor vasculature normalization was associated with decreased permeability and increased perfusion. These changes in vascular function in BMP9-overexpressing LLC tumors resulted in significant alterations of the tumor microenvironment, characterized by a decrease in hypoxia and an increase in immune infiltration. In conclusion, we show that BMP9 promotes vascular normalization in LLC tumors that leads to changes in the microenvironment.

Immature human dendritic cells enhance their migration through KCa3.1 channel activation.

Migration capacity is essential for dendritic cells (DCs) to present antigen to T cells for the induction of immune response. The DC migration is supposed to be a calcium-dependent process, while not fully understood. Here, we report a role of the KCa3.1/IK1/SK4 channels in the migration capacity of both immature (iDC) and mature (mDC) human CD14(+)-derived DCs. KCa3.1 channels were shown to control the membrane potential of human DC and the Ca(2+) entry, which is directly related to migration capacities. The expression of migration marker such as CCR5 and CCR7 was modified in both types of DCs by TRAM-34 (100nM). But, only the migration of iDC was decreased by use of both TRAM-34 and KCa3.1 siRNA. Confocal analyses showed a close localization of CCR5 with KCa3.1 in the steady state of iDC. Finally, the implication of KCa3.1 seems to be limited to the migration capacities as T cell activation of DCs appeared unchanged. Altogether, these results demonstrated that KCa3.1 channels have a pro-migratory effect on iDC migration. Our findings suggest that KCa3.1 in human iDC play a major role in their migration and constitute an attractive target for the cell therapy optimization.