Charles-Antoine Dutertre

Unsupervised High-Dimensional Analysis Aligns Dendritic Cells across Tissues and Species.

Summary Dendritic cells (DCs) are professional antigen-presenting cells that hold great therapeutic potential. Multiple DC subsets have been described, and it remains challenging to align them across tissues and species to analyze their function in the absence of macrophage contamination. Here, we provide and validate a universal toolbox for the automated identification of DCs through unsupervised analysis of conventional flow cytometry and mass cytometry data obtained from multiple mouse, macaque, and human tissues. The use of a minimal set of lineage-imprinted markers was sufficient to subdivide DCs into conventional type 1 (cDC1s), conventional type 2 (cDC2s), and plasmacytoid DCs (pDCs) across tissues and species. This way, a large number of additional markers can still be used to further characterize the heterogeneity of DCs across tissues and during inflammation. This framework represents the way forward to a universal, high-throughput, and standardized analysis of DC populations from mutant mice and human patients.

Mapping the human DC lineage through the integration of high-dimensional techniques

Tracing development of the dendritic cell lineage Dendritic cells (DCs) are important components of the immune system that form from the bone marrow into two major cell lineages: plasmacytoid DCs and conventional DCs. See et al. applied single-cell RNA sequencing and cytometry by time-of-flight to characterize the developmental pathways of these cells. They identified blood DC precursors that shared surface markers with plasmacytoid DCs but that were functionally distinct. This unsuspected level of complexity in pre-DC populations reveals additional cell types and refines understanding of known cell types. Science, this issue p. eaag3009 In human blood, the immunological dendritic cell lineage contains many predendritic cell populations. INTRODUCTION Dendritic cells (DC) are professional antigen-presenting cells that orchestrate immune responses. The human DC population comprises multiple subsets, including plasmacytoid DC (pDC) and two functionally specialized lineages of conventional DC (cDC1 and cDC2), whose origins and differentiation pathways remain incompletely defined. RATIONALE As DC are essential regulators of the immune response in health and disease, potential intervention strategies aiming at manipulation of these cells will require in-depth insights of their origins, the mechanisms that govern their homeostasis, and their functional properties. Here, we employed two unbiased high-dimensional technologies to characterize the human DC lineage from bone marrow to blood. RESULTS We isolated the DC-containing population (Lineage−HLA−DR+CD135+ cells) from human blood and defined the transcriptomes of 710 individual cells using massively parallel single-cell mRNA sequencing. By combining complementary bioinformatic approaches, we identified a small cluster of cells within this population as putative DC precursors (pre-DC). We then confirmed this finding using cytometry by time-of-flight (CyTOF) to simultaneously measure the expression of a panel of 38 different proteins at the single-cell level on Lineage−HLA−DR+ cells and found that pre-DC possessed a CD123+CD33+CD45RA+ phenotype. We confirmed the precursor potential of pre-DC by establishing their potential to differentiate in vitro into cDC1 and cDC2, but not pDC, in the known proportions found in vivo. Interestingly, pre-DC also express classical pDC markers, including CD123, CD303, and CD304. Thus, any previous studies using these markers to identify or isolate pDC will have inadvertently included CD123+CD33+ pre-DC. We provide here new markers that can be used to identify unambiguously pre-DC from pDC, including CD33, CX3CR1, CD2, CD5, and CD327. When CD123+CD33+ pre-DC and CD123+CD33− pDC were isolated separately, we observed that pre-DC have unique functional properties that were previously attributed to pDC. Although pDC remain bona fide interferon-α–producing cells, their reported interleukin-12 (IL-12) production and CD4 T cell allostimulatory capacity can likely be attributed to “contaminating” pre-DC. We then asked whether the pre-DC population contained both uncommitted and committed pre-cDC1 and pre-cDC2 precursors, as recently shown in mice. Using microfluidic single-cell mRNA sequencing (scmRNAseq), we showed that the human pre-DC population contains cells exhibiting transcriptomic priming toward cDC1 and cDC2 lineages. Flow cytometry and in vitro DC differentiation experiments further identified CD123+CADM1−CD1c− putative uncommitted pre-DC, alongside CADM1+CD1c− pre-cDC1 and CADM1−CD1c+ pre-cDC2. Finally, we found that pre-DC subsets expressed T cell costimulatory molecules and induced comparable proliferation and polarization of naïve CD4 T cells as adult DC. However, exposure to the Toll-like receptor 9 (TLR9) ligand CpG triggered IL-12p40 and tumor necrosis factor–α production by early pre-DC, pre-cDC1, and pre-cDC2, in contrast to differentiated cDC1 and cDC2, which do not express TLR9. CONCLUSION Using unsupervised scmRNAseq and CyTOF analyses, we have unraveled the complexity of the human DC lineage at the single-cell level, revealing a continuous process of differentiation that starts in the bone marrow (BM) with common DC progenitors (CDP), diverges at the point of emergence of pre-DC and pDC potential, and culminates in maturation of both lineages in the blood and spleen. The pre-DC compartment contains functionally and phenotypically distinct lineage-committed subpopulations, including one early uncommitted CD123+ pre-DC subset and two CD45RA+CD123lo lineage-committed subsets. The discovery of multiple committed pre-DC populations with unique capabilities opens promising new avenues for the therapeutic exploitation of DC subset-specific targeting. Human DC emerge from BM CDP, diverge at the point of emergence of pre-DC and pDC potential, and culminate in maturation of both lineages in the blood. The pre-DC compartment further differentiates into functionally and phenotypically distinct lineage-committed subpopulations, including one early uncommitted CD123+ pre-DC subset (early pre-DC), which give rise to both cDC1 and cDC2 through corresponding CD45RA+CD123lo pre-cDC1 and pre-cDC2 lineage-committed subsets, respectively. Dendritic cells (DC) are professional antigen-presenting cells that orchestrate immune responses. The human DC population comprises two main functionally specialized lineages, whose origins and differentiation pathways remain incompletely defined. Here, we combine two high-dimensional technologies—single-cell messenger RNA sequencing (scmRNAseq) and cytometry by time-of-flight (CyTOF)—to identify human blood CD123+CD33+CD45RA+ DC precursors (pre-DC). Pre-DC share surface markers with plasmacytoid DC (pDC) but have distinct functional properties that were previously attributed to pDC. Tracing the differentiation of DC from the bone marrow to the peripheral blood revealed that the pre-DC compartment contains distinct lineage-committed subpopulations, including one early uncommitted CD123high pre-DC subset and two CD45RA+CD123low lineage-committed subsets exhibiting functional differences. The discovery of multiple committed pre-DC populations opens promising new avenues for the therapeutic exploitation of DC subset-specific targeting.

Aligning bona fide dendritic cell populations across species

Dendritic cells (DC) are professional antigen sensing and presenting cells that link innate and adaptive immunity. Consisting of functionally specialized subsets, they form a complex cellular network capable of integrating multiple environmental signals leading to immunity or tolerance. Much of DC research so far has been carried out in mice and increasing efforts are now being devoted to translating the findings into humans and other species. Recent studies have aligned these cellular networks across species at multiple levels from phenotype, gene expression program, ontogeny and functional specializations. In this review, we focus on recent advances in the definition of bona fide DC subsets across species. The understanding of functional similarities and differences of specific DC subsets in different animals not only brings light in the field of DC biology, but also paves the way for the design of future effective therapeutic strategies targeting these cells.

Monocyte-derived dendritic cells from HLA-B27+ axial spondyloarthritis (SpA) patients display altered functional capacity and deregulated gene expression

IntroductionThis study aimed to compare the functional capacity and gene expression profile of monocyte-derived dendritic cells (MD-DCs) in HLA-B27+ axial spondyloarthritis (SpA) patients and healthy controls.MethodsMD-DCs were differentiated with interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) for seven days, starting from purified CD14+ monocytes and stimulated with lipopolysaccharide (LPS) for six and twenty four hours. Their capacity to stimulate allogeneic CD4+ T cells from unrelated healthy donor was tested. Transcriptomic study was performed with Affymetrix HuGene 1.0 ST microarrays. Gene expression levels were compared between patients and controls using a multivariate design under a linear model (LIMMA). Real-time quantitative PCR (qRT-PCR) was performed for validation of the most striking gene expression differences.ResultsThe stimulatory capacity of allogeneic CD4+ T cells by MD-DCs from SpA patients was decreased. Transcriptomic analysis revealed 81 genes differentially expressed in MD-DCs between SpA patients and controls (P <0.01 and fold-change <0.66 or >1.5). Four selected genes were validated by qRT-PCR: ADAMTS15, CITED2, F13A1 and SELL. Expression levels of ADAMTS15 and CITED2, encoding a metallopeptidase and a transcription factor, respectively, were inversely correlated with each other (R = 0.75, P = 0.0003). Furthermore, in silico analysis identified several genes of the Wnt signaling pathway having expression co-regulated with CITED2.ConclusionThis study revealed altered function and gene expression pattern in MD-DCs from HLA-B27+ axial SpA. Co-expression study showed an inverse correlation between ADAMTS15 and CITED2. Moreover, the Wnt signaling pathway appeared as deregulated in SpA MD-DCs, a finding which may be connected to Th17-driven inflammatory responses.

Phenotypic and functional characterization of the major lymphocyte populations in the fruit-eating bat Pteropus alecto

The unique ability of bats to act as reservoir for viruses that are highly pathogenic to humans suggests unique properties and functional characteristics of their immune system. However, the lack of bat specific reagents, in particular antibodies, has limited our knowledge of bat’s immunity. Using cross-reactive antibodies, we report the phenotypic and functional characterization of T cell subsets, B and NK cells in the fruit-eating bat Pteropus alecto. Our findings indicate the predominance of CD8+ T cells in the spleen from wild-caught bats that may reflect either the presence of viruses in this organ or predominance of this cell subset at steady state. Instead majority of T cells in circulation, lymph nodes and bone marrow (BM) were CD4+ subsets. Interestingly, 40% of spleen T cells expressed constitutively IL-17, IL-22 and TGF-β mRNA, which may indicate a strong bias towards the Th17 and regulatory T cell subsets. Furthermore, the unexpected high number of T cells in bats BM could suggest an important role in T cell development. Finally, mitogenic stimulation induced proliferation and production of effector molecules by bats immune cells. This work contributes to a better understanding of bat’s immunity, opening up new perspectives of therapeutic interventions for humans.

Unlocking bat immunology: establishment of Pteropus alecto bone marrow-derived dendritic cells and macrophages

Bats carry and shed many emerging infectious disease agents including Ebola virus and SARS-like Coronaviruses, yet they rarely display clinical symptoms of infection. Bat epithelial or fibroblast cell lines were previously established to study the bat immune response against viral infection. However, the lack of professional immune cells such as dendritic cells (DC) and macrophages has greatly limited the significance of current investigations. Using Pteropus alecto (P. alecto) GM-CSF plus IL4, FLT3L and CSF-1, we successfully generated bat bone marrow-derived DC and macrophages. Cells with the phenotype, morphology and functional features of monocyte-derived DC, bona fide DC or macrophages were obtained in GM-CSF/IL4, FLT3L or CSF-1 cultures, respectively. The successful generation of the first bat bone marrow-derived immune cells paves the way to unlocking the immune mechanisms that confer host resilience to pathogens in bats.

Intrahepatic CD206+ macrophages contribute to inflammation in advanced viral-related liver disease

BACKGROUND & AIMSnLiver inflammation is key in the progression of chronic viral hepatitis to cirrhosis and hepatocellular carcinoma. The magnitude of viral replication and the specific anti-viral immune responses should govern the degree of inflammation, but a direct correlation is not consistently found in chronic viral hepatitis patients. We aim to better define the mechanisms that contribute to chronic liver inflammation.nnnMETHODSnIntrahepatic CD14+ myeloid cells from healthy donors (n=19) and patients with viral-related liver cirrhosis (HBV, HBV/HDV or HCV; n=15) were subjected to detailed phenotypic, molecular and functional characterisation.nnnRESULTSnUnsupervised analysis of multi-parametric data showed that liver disease was associated with the intrahepatic expansion of activated myeloid cells mainly composed of pro-inflammatory CD14+HLA-DRhiCD206+ cells, which spontaneously produced TNFα and GM-CSF. These cells only showed heightened pro-inflammatory responses to bacterial TLR agonists and were more refractory to endotoxin-induced tolerance. A liver-specific enrichment of CD14+HLA-DRhiCD206+ cells was also detected in a humanised mouse model of liver inflammation. This accumulation was abrogated following oral antibiotic treatment, suggesting a direct involvement of translocated gut-derived microbial products in liver injury.nnnCONCLUSIONSnViral-related chronic liver inflammation is driven by the interplay between non-endotoxin-tolerant pro-inflammatory CD14+HLA-DRhiCD206+ myeloid cells and translocated bacterial products. Deciphering this mechanism paves the way for the development of therapeutic strategies specifically targeting CD206+ myeloid cells in viral-related liver disease patients. Lay summary: Viral-related chronic liver disease is driven by intrahepatic pro-inflammatory myeloid cells accumulating in a gut-derived bacterial product-dependent manner. Our findings support the use of oral antibiotics to ameliorate liver inflammation in these patients.

Bat-mouse bone marrow chimera: a novel animal model for dissecting the uniqueness of the bat immune system.

Bats are an important animal model with long lifespans, low incidences of tumorigenesis and an ability to asymptomatically harbour pathogens. Currently, in vivo studies of bats are hampered due to their low reproduction rates. To overcome this, we transplanted bat cells from bone marrow (BM) and spleen into an immunodeficient mouse strain NOD-scid IL-2R−/− (NSG), and have successfully established stable, long-term reconstitution of bat immune cells in mice (bat-mice). Immune functionality of our bat-mouse model was demonstrated through generation of antigen-specific antibody response by bat cells following immunization. Post-engraftment of total bat BM cells and splenocytes, bat immune cells survived, expanded and repopulated the mouse without any observable clinical abnormalities. Utilizing bat’s remarkable immunological functions, this novel model has a potential to be transformed into a powerful platform for basic and translational research.

Identification of human DC precursors through the integration of high dimensional strategies

CD4+Foxp3+ regulatory T cells (Tregs) are the main regulators of peripheral tolerance and prevent the development of fatal autoimmune disease in humans and mice. Furthermore, Tregs have also been implicated in suppressing anti-tumour immune responses and are often enriched at nsites of primary and metastatic tumours. While studies have shown the effect of Treg ablation on the control of primary tumours, few studies have examined their contribution to metastasis progression. nIn this thesis I hypothesised that the depletion of Tregs could promote control over metastasis. To address this, a highly metastatic murine mammary carcinoma cell line 4T1 was injected into transgenic mice expressing the diphtheria toxin receptor in Foxp3+ cells. Foxp3+ cells were depleted by administration of diphtheria toxin and the impact of this on growth of primary tumours and metastases was assessed and measured in vitro clonogenic assays. Results of these experiments indicated that Tregdepletion nled to control of primary tumour growth and in some mice to control of metastases. Control of metastases was linked to control of primary tumour growth. nIn order to measure metastasis in vivo, a PET/CT imaging technique was optimized. Primary tumours and large metastatic nodules were successfully imaged in mice using F18 FDG as a radiotracer. However, the studies described herein revealed that micrometastases in mouse lungs nwere too small to be reliably identified using PET data parameters. CT imaging did however enable detection of increases in tissue density within the lungs, which was suggestive of micrometastases. Data obtained in this way also indicated that Treg-depletion promotes control of metastasis in some mice. nCollectively, the findings described in this thesis indicate that Tregdepletion can contribute to control of metastatic disease and should therefore represent an important component of novel immunotherapies.s of ICI 2016 International Congress of Immunology 21-26 August 2016 Melbourne, Australia European Journal of Immunology Volume 46, Suppl. 1, August 2016 This abstract book can be searched using the PDF search function to look, for example, for the Abstract number or author name. To cite an Abstract, please use the following format: Abstract title. Authors. Conference: ICI 2016. Location Melbourne, Australia. Date Aug 2126, 2016. Eur. J. Immunol. 2016. 46, S1, page number(s). Meeting Abstract number [thetitle. Authors. Conference: ICI 2016. Location Melbourne, Australia. Date Aug 2126, 2016. Eur. J. Immunol. 2016. 46, S1, page number(s). Meeting Abstract number [the Abstract number can be found above the title]number can be found above the title]Changes in microbiome, mucosal immunity and intestinal integrity have been associated with the onset of Type 1 Diabetes (T1D) in children. Toll-like Receptors (TLR) have been associated all three factors. The role of TLR and their effects on microbiome in autoimmunity were studied by crossing TLR1,2,4,6,9 and MyD88 targeted deficiency mutations to the type 1 diabetes (T1D)-prone NOD mouse strain. While NOD.Tlr9-/- and NOD.Tlr6-/- mice were unaffected, T1D in NOD.Tlr4-/- and NOD.Tlr1-/- mice was exacerbated and that in NOD.Myd88-/- and NOD.Tlr2-/- mice ameliorated. Physical parameters of the intestines were compared; ileal weight was reduced in NOD.Tlr1-/-mice. Similarly, by histology, these mice had reduced villus length and width. The intestinal microbiomes of NOD wild-type (WT), NOD.Tlr1-/-, NOD.Tlr2-/- and NOD.Tlr4-/- mice were compared by high throughput sequencing of 16S ribosomal DNA (rDNA), in two cohorts, 18 months apart. Analysis of caecal 16S sequences clearly resolved the mouse lines and there were significant differences in beta diversity between the strains, with individual bacterial species contributing greatly to the differences in the microbiota of individual TLR-deficient strains. To test the relationship between microbiome and T1D, all strains were re-derived onto the parental NOD/Lt line and the incidence of T1D re-assessed within two generations. All rederived lines expressed an incidence of disease similar to that of the parental line. TLR deficiencies are associated with changes in microbiome; changes of microbiome are associated with T1D; the effects of TLR deficiencies on T1D appear to be mediated by their effects on gut flora.Intestinal TCRb+CD4-CD8b-CD8a+ (CD8aa) IELs alleviate T cell induced colitis and have been suggested to play a role in virus infection and cancer. Their thymic development has been elucidated to some extent, as IEL precursors (IELp) are known to be CD4-CD8-CD5+TCRb+, but is not yet fully understood. Within the thymus, mature IELp were identified based on their expression of CD122 and MHC class I. Two major phenotypic subsets exist within this mature thymic IELp population: a PD1+Tbet- population that preferentially expresses a4b7, and a PD1-Tbet+ population with preferential CD103 expression. These two populations were also distinct in their Valpha repertoire. The PD1+a4b7+ population contains clones that are strongly self-reactive as judged by Nur77GFP and their dramatic increase in Bim deficient mice, while the PD1-Tbet+ population did not show these characteristics. Both gave rise to CD8aa IELs upon adoptive transfer into RAG-/- recipients. However intrathymic labeling revealed that PD1+a4b7+ IELp were the major thymic emigrating population, and emigration was S1P1-dependent. In contrast, PD1-Tbet+ IELp expressed CXCR3, were retained, and accumulated in the thymus with age. Preliminary immunofluorescence data furthermore indicate differential thymic cortico-medullary localization of the IELp subtypes. These experiments more precisely define the behavior of IEL precursors.Multiple Sclerosis (MS), the most common disabling neurological disease affecting young adults in developed countries, is a complex genetic disease associated with both environmental and genetic risk factors. In most cases, the risk factors individual associations with MS are so weak that any meaningful understanding of the disease will require the identification of molecular pathways that contribute to MS liability. We therefore hypothesisedthat the complex genetic phenotype is driven by a co-ordinated expression of transcriptional regulatory networks. To test this, we generated a weighted gene co-expression network based on 712 pooled Affymetrix Human Gene 1.0 ST array analyses of magnetic bead sorted B cells, CD4 and CD8 T cells, NK cells and monocytes, from 67 untreated relapsing/remitting MS patients and 102 Healthy Controls (HC). Sixteen relatively independent gene modules were identified. For each leukocyte population, the strength of differential expression between patients and HC was assessed, by ranking genes by Mann Whitney U test and ANOVA, and each transcript was tested across the network to identify modules of interest. A group of transcripts we named the “Black” module was most significantly associated with MS in monocytes & was strongly down-regulated in patients. Twelve highly differentially expressed genes with high centrality were identified and the top annotation clusters comprised the immune processes: Natural killer cell mediated cytotoxicity & Antigen processing and presentation. We propose that manipulating the module as a whole may provide a new perspective on the aetiology of complex genetic diseases and offer novel therapies for MS.