Single-Cell Analyses Inform Mechanisms of Myeloid-Targeted Therapies in Colon Cancer.

Single-cell RNA sequencing (scRNA-seq) is a powerful tool for defining cellular diversity in tumors, but its application toward dissecting mechanisms underlying immune-modulating therapies is scarce. We performed scRNA-seq analyses on immune and stromal populations from colorectal cancer patients, identifying specific macrophage and conventional dendritic cell (cDC) subsets as key mediators of cellular cross-talk in the tumor microenvironment. Defining comparable myeloid populations in mouse tumors enabled characterization of their response to myeloid-targeted immunotherapy. Treatment with anti-CSF1R preferentially depleted macrophages with an inflammatory signature but spared macrophage populations that in mouse and human expresses pro-angiogenic/tumorigenic genes. Treatment with a CD40 agonist antibody preferentially activated a cDC population and increased Bhlhe40+ Th1-like cells and CD8+ memory T cells. Our comprehensive analysis of key myeloid subsets in human and mouse identifies critical cellular interactions regulating tumor immunity and defines mechanisms underlying myeloid-targeted immunotherapies currently undergoing clinical testing.

Transcriptional and Functional Analysis of CD1c+ Human Dendritic Cells Identifies a CD163+ Subset Priming CD8+CD103+ T Cells.

Dendritic cells (DCs) are antigen-presenting cells controlling T cell activation. In humans, the diversity, ontogeny, and functional capabilities of DC subsets are not fully understood. Here, we identified circulating CD88-CD1c+CD163+ DCs (called DC3s) as immediate precursors of inflammatory CD88-CD14+CD1c+CD163+FcεRI+ DCs. DC3s develop via a specific pathway activated by GM-CSF, independent of cDC-restricted (CDP) and monocyte-restricted (cMoP) progenitors. Like classical DCs but unlike monocytes, DC3s drove activation of naive T cells. In vitro, DC3s displayed a distinctive ability to prime CD8+ T cells expressing a tissue homing signature and the epithelial homing alpha-E integrin (CD103) through transforming growth factor ß (TGF-ß) signaling. In vivo, DC3s infiltrated luminal breast cancer primary tumors, and DC3 infiltration correlated positively with CD8+CD103+CD69+ tissue-resident memory T cells. Together, these findings define DC3s as a lineage of inflammatory DCs endowed with a strong potential to regulate tumor immunity.

The role of dendritic cells in the instruction of helper T cells in the allergic march.

Allergy is a complex array of diseases influenced by innate and adaptive immunity, genetic polymorphisms, and environmental triggers. Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by barrier defects and immune dysregulation, sometimes leading to asthma and food allergies because of the atopic march. During atopic skin inflammation, Langerhans cells and dendritic cells (DCs) in the skin capture and deliver allergen information to local lymph nodes. DCs are essential immune sensors coordinating immune reactions by capturing and presenting antigens to T cells. In the context of allergic responses, DCs play a crucial role in instructing two types of helper T cells - type 2 helper T (Th2) cells and follicular helper T (TFH) cells - in allergic responses and IgE antibody responses. In skin sensitization, the differentiation and function of Th2 cells and TFH cells are influenced by skin-derived factors, including epithelial cytokines, chemokines, and signaling pathways to modify the function of migratory DCs and conventional DCs. In this review, we aim to understand the specific mechanisms involving DCs in allergic responses to provide insights into the pathogenesis of allergic diseases and potential therapeutic strategies.

Attenuation of renal injury by depleting cDC1 and by repurposing Flt3 inhibitor in anti-GBM disease.

Previous studies found cDC1s to be protective in early stage anti-GBM disease through Tregs, but pathogenic in late stage Adriamycin nephropathy through CD8+ T cells. Flt3 ligand is a growth factor essential for cDC1 development and Flt3 inhibitors are currently used for cancer treatment. We conducted this study to clarify the role and mechanisms of effects of cDC1s at different time points in anti-GBM disease. In addition, we aimed to utilize drug repurposing of Flt3 inhibitors to target cDC1s as a treatment of anti-GBM disease. We found that in human anti-GBM disease, the number of cDC1s increased significantly, proportionally more than cDC2s. The number of CD8+ T cells also increased significantly and their number correlated with cDC1 number. In XCR1-DTR mice, late (day 12-21) but not early (day 3-12) depletion of cDC1s attenuated kidney injury in mice with anti-GBM disease. cDC1s separated from kidneys of anti-GBM disease mice were found to have a pro-inflammatory phenotype (i.e. express high level of IL-6, IL-12 and IL-23) in late but not early stage. In the late depletion model, the number of CD8+ T cells was also reduced, but not Tregs. CD8+ T cells separated from kidneys of anti-GBM disease mice expressed high levels of cytotoxic molecules (granzyme B and perforin) and inflammatory cytokines (TNF-α and IFN-γ), and their expression reduced significantly after cDC1 depletion with diphtheria toxin. These findings were reproduced using a Flt3 inhibitor in wild type mice. Therefore, cDC1s are pathogenic in anti-GBM disease through activation of CD8+ T cells. Flt3 inhibition successfully attenuated kidney injury through depletion of cDC1s. Repurposing Flt3 inhibitors has potential as a novel therapeutic strategy for anti-GBM disease.

Comprehensive Phenotyping of Dendritic Cells in Cancer Patients by Flow Cytometry.

Dendritic cells (DCs) play a crucial role in the complex interplay between tumor cells and the immune system. During the elimination phase of cancer immunoediting, immunostimulatory DCs are critical for the control of tumor growth. During the escape phase, regulatory DCs sustain tumor tolerance and contribute to the development of the immunosuppressive tumor microenvironment that characterizes this phase. Moreover, increasing evidence indicates that DCs are also critical for the success of cancer immunotherapy. Hence, there is increasing need to fully characterize DC subsets and their activatory/inhibitory profile in cancer patients. In this review, we describe the role played by different DC subsets in the different phases of cancer immunoediting, the function exerted by different activatory and inhibitory molecules expressed on DC surface, and the cytokines produced by distinct DC subsets, in order to provide an overview on the DC features that may be useful to be assessed when dealing with the flow cytometric characterization of DCs in cancer patients. © 2020 International Society for Advancement of Cytometry.

Autoimmune animal models in the analysis of the CD47-SIRPα signaling pathway.

Signal regulatory protein α (SIRPα), also known as SHPS-1/SIRPA, is an immunoglobulin superfamily protein that binds to the protein tyrosine phosphatases Shp1 and Shp2 through its cytoplasmic region and is predominantly expressed in dendritic cells and macrophages. CD47, a widely expressed transmembrane protein, is a ligand for SIRPα, with the two proteins constituting a cell-cell communication system. It was previously demonstrated that the CD47-SIRPα signaling pathway is important for prevention of clearance by splenic macrophages of red blood cells or platelets from the bloodstream. In addition, this signaling pathway is also implicated in homeostatic regulation of dendritic cells and development of autoimmunity. Here we describe the detailed protocols for methods that were used in our recent studies to study the role of the CD47-SIRPα signaling pathway in autoimmunity. We also demonstrate that hematopoietic SIRPα as well as nonhematopoietic CD47 are important for development of experimental autoimmune encephalomyelitis. Thus, we here strengthen the importance of experimental animal models as well as other methods for the study of molecular pathogenesis of autoimmunity.

Inhibition of CSF-1R and IL-6R prevents conversion of cDC2s into immune incompetent tumor-induced DC3s boosting DC-driven therapy potential.

The human dendritic cell (DC) family has recently been expanded by CD1c+CD14+CD163+ DCs, introduced as DC3s. DC3s are found in tumors and peripheral blood of cancer patients. Here, we report elevated frequencies of CD14+ cDC2s, which restore to normal frequencies after tumor resection, in non-small cell lung cancer patients. These CD14+ cDC2s phenotypically resemble DC3s and exhibit increased PD-L1, MERTK, IL-10, and IDO expression, consistent with inferior T cell activation ability compared with CD14- cDC2s. In melanoma patients undergoing CD1c+ DC vaccinations, increased CD1c+CD14+ DC frequencies correlate with reduced survival. We demonstrate conversion of CD5+/-CD1c+CD14- cDC2s to CD14+ cDC2s by tumor-associated factors, whereas monocytes failed to express CD1c under similar conditions. Targeted proteomics identified IL-6 and M-CSF as dominant drivers, and we show that IL-6R and CSF1R inhibition prevents tumor-induced CD14+ cDC2s. Together, this indicates cDC2s as direct pre-cursors of DC3-like CD1c+CD14+ DCs and provides insights into the importance and modulation of CD14+ DC3s in anti-tumor immune responses.

The Th1/Tfh-like biased responses elicited by the rASP-1 innate adjuvant are dependent on TRIF and Type I IFN receptor pathways.

Ov-ASP-1 (rASP-1), a parasite-derived protein secreted by the helminth Onchocerca volvulus, is an adjuvant which enhances the potency of the influenza trivalent vaccine (IIV3), even when used with 40-fold less IIV3. This study is aimed to provide a deeper insight into the molecular networks that underline the adjuvanticity of rASP-1. Here we show that rASP-1 stimulates mouse CD11c+ bone marrow-derived dendritic (BMDCs) to secrete elevated levels of IL-12p40, TNF-α, IP-10 and IFN-ß in a TRIF-dependent but MyD88-independent manner. rASP-1-activated BMDCs promoted the differentiation of naïve CD4+ T cells into Th1 cells (IFN-γ+) that was TRIF- and type I interferon receptor (IFNAR)-dependent, and into Tfh-like cells (IL21+) and Tfh1 (IFN-γ+ IL21+) that were TRIF-, MyD88- and IFNAR-dependent. rASP-1-activated BMDCs promoted the differentiation of naïve CD4+ T cells into Th17 (IL-17+) cells only when the MyD88 pathway was inhibited. Importantly, rASP-1-activated human blood cDCs expressed upregulated genes that are associated with DC maturation, type I IFN and type II IFN signaling, as well as TLR4-TRIF dependent signaling. These activated cDCs promoted the differentiation of naïve human CD4+ T cells into Th1, Tfh-like and Th17 cells. Our data thus confirms that the rASP-1 is a potent innate adjuvant that polarizes the adaptive T cell responses to Th1/Tfh1 in both mouse and human DCs. Notably, the rASP-1-adjuvanted IIV3 vaccine elicited protection of mice from a lethal H1N1 infection that is also dependent on the TLR4-TRIF axis and IFNAR signaling pathway, as well as on its ability to induce anti-IIV3 antibody production.

Conventional Type 1 Dendritic Cells (cDC1) in Human Kidney Diseases: Clinico-Pathological Correlations.

Background: cDC1 is a subset of conventional DCs, whose most recognized function is cross-presentation to CD8+ T cells. We conducted this study to investigate the number and location of cDC1s in various human kidney diseases as well as their correlation with clinico-pathological features and CD8+ T cells. Methods: We analyzed 135 kidney biopsies samples. Kidney diseases included: acute tubular necrosis (ATN), acute interstitial nephritis (AIN), proliferative glomerulonephritis (GN) (IgA nephropathy, lupus nephritis, pauci-immune GN, anti-GBM disease), non-proliferative GN (minimal change disease, membranous nephropathy) and diabetic nephropathy. Indirect immunofluorescence staining was used to quantify cDC1s, CD1c+ DCs, and CD8+ T cells. Results: cDC1s were rarely present in normal kidneys. Their number increased significantly in ATN and proliferative GN, proportionally much more than CD1c+ DCs. cDC1s were mainly found in the interstitium, except in lupus nephritis, pauci-immune GN and anti-GBM disease, where they were prominent in glomeruli and peri-glomerular regions. The number of cDC1s correlated with disease severity in ATN, number of crescents in pauci-immune GN, interstitial fibrosis in IgA nephropathy and lupus nephritis, as well as prognosis in IgA nephropathy. The number of CD8+ T cells also increased significantly in these conditions and cDC1 number correlated with CD8+ T cell number in lupus nephritis and pauci-immune GN, with many of them closely co-localized. Conclusions: cDC1 number correlated with various clinic-pathological features and prognosis reflecting a possible role in these conditions. Their association with CD8+ T cells suggests a combined mechanism in keeping with the results in animal models.

Human Dendritic Cells: Ontogeny and Their Subsets in Health and Disease.

Dendritic cells (DCs) are a type of cells derived from bone marrow that represent 1% or less of the total hematopoietic cells of any lymphoid organ or of the total cell count of the blood or epithelia. Dendritic cells comprise a heterogeneous population of cells localized in different tissues where they act as sentinels continuously capturing antigens to present them to T cells. Dendritic cells are uniquely capable of attracting and activating naïve CD4⁺ and CD8⁺ T cells to initiate and modulate primary immune responses. They have the ability to coordinate tolerance or immunity depending on their activation status, which is why they are also considered as the orchestrating cells of the immune response. The purpose of this review is to provide a general overview of the current knowledge on ontogeny and subsets of human dendritic cells as well as their function and different biological roles.

The Isolation and Enrichment of Large Numbers of Highly Purified Mouse Spleen Dendritic Cell Populations and Their In Vitro Equivalents.

Dendritic cells (DCs) form a complex network of cells that initiate and orchestrate immune responses against a vast array of pathogenic challenges. Developmentally and functionally distinct DC subtypes differentially regulate T-cell function. Importantly it is the ability of DC to capture and process antigen, whether from pathogens, vaccines, or self-components, and present it to naive T cells that is the key to their ability to initiate an immune response. Our typical isolation procedure for DC from murine spleen was designed to efficiently extract all DC subtypes, without bias and without alteration to their in vivo phenotype, and involves a short collagenase digestion of the tissue, followed by selection for cells of light density and finally negative selection for DC. The isolation procedure can accommodate DC numbers that have been artificially increased via administration of fms-like tyrosine kinase 3 ligand (Flt3L), either directly through a series of subcutaneous injections or by seeding with an Flt3L secreting murine melanoma. Flt3L may also be added to bone marrow cultures to produce large numbers of in vitro equivalents of the spleen DC subsets. Total DC, or their subsets, may be further purified using immunofluorescent labeling and flow cytometric cell sorting. Cell sorting may be completely bypassed by separating DC subsets using a combination of fluorescent antibody labeling and anti-fluorochrome magnetic beads. Our procedure enables efficient separation of the distinct DC subsets, even in cases where mouse numbers or flow cytometric cell sorting time is limiting.

Dendritic cells generated with Flt3L and exposed to apoptotic cells lack induction of T cell anergy and Foxp3⁺ regulatory T cell conversion in vitro.

Removal of apoptotic cells, which appear during the steady state, is a pre-requisite to prevent generation of secondary necrotic cells that may lead to autoimmunity. The recognition of apoptotic material by dendritic cells (DCs) has been proposed to convert them into tolerogenic DCs equipped with specialized tolerogenic mechanisms on T cells. However, comparative studies to demonstrate functional alterations of DCs upon exposure to apoptotic cells have not been performed so far. Here we show that immature murine bone marrow-derived DCs generated with GM-CSF (GM-DCs) or Flt3L (FL-DCs) interact with live or apoptotic syngeneic thymocytes. As expected, GM-DCs phagocytose apoptotic but not live cells, FL-DCs only show trogocytosis of membrane parts. Interaction with live or apoptotic thymocytes did not lead to DC maturation. Both GM-DCs and FL-DCs present OVA as protein, peptide and membrane-associated antigens. Interestingly, only GM-DCs were able to induce T cell anergy or convert naïve T cells into FoxP3⁺ regulatory T cells (Tregs) but FL-DCs did not show either of these effects. Unexpectedly, exposure of immature GM-DCs to live or apoptotic thymocytes did not improve DC functions in both types of in vitro T cell tolerance induction assays. Together, our data suggest that these tolerogenic in vitro measures of immature BM-DCs are not further enhanced by exposure to apoptotic cells and may depend on the generating cytokine.