Mitochondrial control of microglial phagocytosis by the translocator protein and hexokinase 2 in Alzheimer's disease.

Microglial phagocytosis is an energetically demanding process that plays a critical role in the removal of toxic protein aggregates in Alzheimer's disease (AD). Recent evidence indicates that a switch in energy production from mitochondrial respiration to glycolysis disrupts this important protective microglial function and may provide therapeutic targets for AD. Here, we demonstrate that the translocator protein (TSPO) and a member of its mitochondrial complex, hexokinase-2 (HK), play critical roles in microglial respiratory-glycolytic metabolism and phagocytosis. Pharmacological and genetic loss-of-function experiments showed that TSPO is critical for microglial respiratory metabolism and energy supply for phagocytosis, and its expression is enriched in phagocytic microglia of AD mice. Meanwhile, HK controlled glycolytic metabolism and phagocytosis via mitochondrial binding or displacement. In cultured microglia, TSPO deletion impaired mitochondrial respiration and increased mitochondrial recruitment of HK, inducing a switch to glycolysis and reducing phagocytosis. To determine the functional significance of mitochondrial HK recruitment, we developed an optogenetic tool for reversible control of HK localization. Displacement of mitochondrial HK inhibited glycolysis and improved phagocytosis in TSPO-knockout microglia. Mitochondrial HK recruitment also coordinated the inflammatory switch to glycolysis that occurs in response to lipopolysaccharide in normal microglia. Interestingly, cytosolic HK increased phagocytosis independent of its metabolic activity, indicating an immune signaling function. Alzheimer's beta amyloid drastically stimulated mitochondrial HK recruitment in cultured microglia, which may contribute to microglial dysfunction in AD. Thus, targeting mitochondrial HK may offer an immunotherapeutic approach to promote phagocytic microglial function in AD.

Turnover Kinetics of Pancreatic Macrophages in Lean and Obese Diabetic Mice.

Pancreatic resident macrophages, a heterogeneous family of cells with distinct origins and phenotypes, are the main myeloid cells in exocrine and endocrine tissues. Adult exocrine F4/80hi macrophages consist of three different subsets based on the embryonic marker Tim-4 and MHC II expression. Their frequencies shift during aging and obesity with the Tim-4-MHCII+ fraction becoming the predominant subpopulation in the inter acinar stroma. Endocrine resident F4/80hi macrophages are more homogenous and represent the prevalent leukocyte fraction residing within the islets in both lean and obese mice. We used an adult fate mapping mouse model to characterize turnover kinetics within the pancreatic resident macrophages under normal homeostasis and obese diabetic conditions. We demonstrate that islet resident macrophages show unique replenishment kinetics, with embryonic macrophages being gradually replaced by bone marrow-derived monocytes with increasing age. Their replenishment was independent of the CCL2/CCR2 axis. Furthermore, we confirmed that both exocrine Tim-4+MHCIIlow and Tim-4+MHCII+ fractions are long-lived and primarily independent from bone marrow-derived monocytes. In contrast, exocrine Tim-4-MHCII+ macrophages are gradually replaced through a CCR2-dependent influx of bone marrow-derived monocytes in aging. Moreover, we show that obesity and type 2 diabetes do not affect the turnover kinetics of any macrophage subpopulation residing in the pancreas. Our study uncovers new insights on pancreatic macrophage biology in aging and obesity.

Intranasal Delivery of RIG-I Agonist Drives Pulmonary Myeloid Cell Activation in Mice.

Viral respiratory infections cause substantial health and economic burden. There is a pressing demand for efficacious vaccination strategies and, therefore, a need for a better understanding of the mechanisms of action of novel potential adjuvants. Here we investigated the effect of a synthetic RIG-I agonist, the dsRNA hairpin 3p10LA9, on the activation of pulmonary myeloid cells. Analysis of early innate immune responses revealed that a single intranasal 3p10LA9 dose induces a transient pulmonary interferon-stimulated gene (ISG) and pro-inflammatory cytokine/chemokine response, which leads to the maturation of three distinct dendritic cell subpopulations in the lungs. While lung resident dendritic cell decrease shortly after 3p10LA9 delivery, their numbers increase in the draining mediastinal lymph node, where they have migrated, maintaining their activated phenotype. At the same time, dsRNA hairpin-induced chemokines attract transiently infiltrating monocytes into the lungs, which causes a short temporary pulmonary inflammation. However, these monocytes are dispensable in controlling influenza infection since in CCR2 deficient mice, lacking these infiltrating cells, the virus load was similar to the wild type mice when infected with the influenza virus at a sublethal dose. In summary, our data suggest that intranasal delivery of dsRNA hairpins, used as a RIG-I targeting adjuvant, represents an attractive strategy to boost type I inteferon-mediated lung dendritic cell maturation, which supports viral reduction in the lungs during infection.

The aryl hydrocarbon receptor instructs the immunomodulatory profile of a subset of Clec4a4+ eosinophils unique to the small intestine.

C-type lectin domain family 4, member a4 (Clec4a4) is a C-type lectin inhibitory receptor specific for glycans thought to be exclusively expressed on murine CD8α− conventional dendritic cells. Using newly generated Clec4a4-mCherry knock-in mice, we identify a subset of Clec4a4-expressing eosinophils uniquely localized in the small intestine lamina propria. Clec4a4+ eosinophils evinced an immunomodulatory signature, whereas Clec4a4− eosinophils manifested a proinflammatory profile. Clec4a4+ eosinophils expressed high levels of aryl hydrocarbon receptor (Ahr), which drove the expression of Clec4a4 as well as other immunomodulatory features, such as PD-L1. The abundance of Clec4a4+ eosinophils was dependent on dietary AHR ligands, increased with aging, and declined in inflammatory conditions. Mice lacking AHR in eosinophils expanded innate lymphoid cells of type 2 and cleared Nippostrongylus brasiliensis infection more effectively than did wild-type mice. These results highlight the heterogeneity of eosinophils in response to tissue cues and identify a unique AHR-dependent subset of eosinophils in the small intestine with an immunomodulatory profile.

Transitional premonocytes emerge in the periphery for host defense against bacterial infections.

Circulating Ly6Chi monocytes often undergo cellular death upon exhaustion of their antibacterial effector functions, which limits their capacity for subsequent macrophage differentiation. This shrouds the understanding on how the host replaces the tissue-resident macrophage niche effectively during bacterial invasion to avert infection morbidity. Here, we show that proliferating transitional premonocytes (TpMos), an immediate precursor of mature Ly6Chi monocytes (MatMos), were mobilized into the periphery in response to acute bacterial infection and sepsis. TpMos were less susceptible to apoptosis and served as the main source of macrophage replenishment when MatMos were vulnerable toward bacteria-induced cellular death. Furthermore, TpMo and its derived macrophages contributed to host defense by balancing the proinflammatory cytokine response of MatMos. Consequently, adoptive transfer of TpMos improved the survival outcome of lethal sepsis. Our findings hence highlight a protective role for TpMos during bacterial infections and their contribution toward monocyte-derived macrophage heterogeneity in distinct disease outcomes.

Microbiota regulates the turnover kinetics of gut macrophages in health and inflammation.

The gut immune system has evolved to co-exist in a mutually beneficial symbiotic relationship with its microflora. Here, using a germ-free fate-mapping mouse model, we provide clear insight into how the enteric commensals determine the kinetics of macrophage turnover. The microbiome density along the gastrointestinal tract defines the persistence of ontogenically diverse macrophages, with the highest numbers of the long-lived F4/80hiTim4+ macrophage subset in the less densely colonized small intestine. Furthermore, the microbiome contributes to a tightly regulated monocyte-dependent replenishment of both long- and short-lived F4/80hi macrophages under homeostatic and inflammatory conditions. In the latter situation, the commensals regulate rapid replenishment of the depleted macrophage niche caused by the intestinal inflammation. The microbial ecosystem imprints a favorable cytokine microenvironment in the intestine to support macrophage survival and monocyte-dependent replenishment. Therefore, the host immune system-commensal cross-talk provides an efficient strategy to assure intestinal homeostasis.

Microglia and CD206+ border-associated mouse macrophages maintain their embryonic origin during Alzheimer's disease.

Brain microglia and border-associated macrophages (BAMs) display distinct spatial, developmental, and phenotypic features. Although at steady state, the origins of distinct brain macrophages are well-documented, the dynamics of their replenishment in neurodegenerative disorders remain elusive, particularly for activated CD11c+ microglia and BAMs. In this study, we conducted a comprehensive fate-mapping analysis of murine microglia and BAMs and their turnover kinetics during Alzheimer's disease (AD) progression. We used a novel inducible AD mouse model to investigate the contribution of bone marrow (BM) cells to the pool of fetal-derived brain macrophages during the development of AD. We demonstrated that microglia remain a remarkably stable embryonic-derived population even during the progression of AD pathology, indicating that neither parenchymal macrophage subpopulation originates from, nor is replenished by, BM-derived cells. At the border-associated brain regions, bona fide CD206+ BAMs are minimally replaced by BM-derived cells, and their turnover rates are not accelerated by AD. In contrast, all other myeloid cells are swiftly replenished by BM progenitors. This information further elucidates the turnover kinetics of these cells not only at steady state, but also in neurodegenerative diseases, which is crucial for identifying potential novel therapeutic targets.

Resident macrophages restrain pathological adipose tissue remodeling and protect vascular integrity in obese mice.

Tissue-resident macrophages in white adipose tissue (WAT) dynamically adapt to the metabolic changes of their microenvironment that are often induced by excess energy intake. Currently, the exact contribution of these macrophages in obesity-driven WAT remodeling remains controversial. Here, using a transgenic CD169-DTR mouse strain, we provide new insights into the interplay between CD169+ adipose tissue macrophages (ATMs) and their surrounding WAT microenvironment. Using targeted in vivo ATM ablation followed by transcriptional and metabolic WAT profiling, we found that ATMs protect WAT from the excessive pathological remodeling that occurs during obesity. As obesity progresses, ATMs control not only vascular integrity, adipocyte function, and lipid and metabolic derangements but also extracellular matrix accumulation and resultant fibrosis in the WAT. The protective role of ATMs during obesity-driven WAT dysfunction supports the notion that ATMs represent friends, rather than foes, as has previously assumed.

Fate mapping analysis reveals a novel murine dermal migratory Langerhans-like cell population.

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.

Renal CD169++ resident macrophages are crucial for protection against acute systemic candidiasis.

Disseminated candidiasis remains as the most common hospital-acquired bloodstream fungal infection with up to 40% mortality rate despite the advancement of medical and hygienic practices. While it is well established that this infection heavily relies on the innate immune response for host survival, much less is known for the protective role elicited by the tissue-resident macrophage (TRM) subsets in the kidney, the prime organ for Candida persistence. Here, we describe a unique CD169++ TRM subset that controls Candida growth and inflammation during acute systemic candidiasis. Their absence causes severe fungal-mediated renal pathology. CD169++ TRMs, without being actively involved in direct fungal clearance, increase host resistance by promoting IFN-γ release and neutrophil ROS activity.

Group 3 Innate Lymphoid Cells Program a Distinct Subset of IL-22BP-Producing Dendritic Cells Demarcating Solitary Intestinal Lymphoid Tissues.

Solitary intestinal lymphoid tissues such as cryptopatches (CPs) and isolated lymphoid follicles (ILFs) constitute steady-state activation hubs containing group 3 innate lymphoid cells (ILC3) that continuously produce interleukin (IL)-22. The outer surface of CPs and ILFs is demarcated by a poorly characterized population of CD11c+ cells. Using genome-wide single-cell transcriptional profiling of intestinal mononuclear phagocytes and multidimensional flow cytometry, we found that CP- and ILF-associated CD11c+ cells were a transcriptionally distinct subset of intestinal cDCs, which we term CIA-DCs. CIA-DCs required programming by CP- and ILF-resident CCR6+ ILC3 via lymphotoxin-ß receptor signaling in cDCs. CIA-DCs differentially expressed genes associated with immunoregulation and were the major cellular source of IL-22 binding protein (IL-22BP) at steady state. Mice lacking CIA-DC-derived IL-22BP exhibited diminished expression of epithelial lipid transporters, reduced lipid resorption, and changes in body fat homeostasis. Our findings provide insight into the design principles of an immunoregulatory checkpoint controlling nutrient absorption.

A Multifunctional Role of Leucine-Rich α-2-Glycoprotein 1 in Cutaneous Wound Healing Under Normal and Diabetic Conditions.

Delayed wound healing is commonly associated with diabetes. It may lead to amputation and death if not treated in a timely fashion. Limited treatments are available partially due to the poor understanding of the complex disease pathophysiology. Here, we investigated the role of leucine-rich α-2-glycoprotein 1 (LRG1) in normal and diabetic wound healing. First, our data showed that LRG1 was significantly increased at the inflammation stage of murine wound healing, and bone marrow-derived cells served as a major source of LRG1. LRG1 deletion causes impaired immune cell infiltration, reepithelialization, and angiogenesis. As a consequence, there is a significant delay in wound closure. On the other hand, LRG1 was markedly induced in diabetic wounds in both humans and mice. LRG1-deficient mice were resistant to diabetes-induced delay in wound repair. We further demonstrated that this could be explained by the mitigation of increased neutrophil extracellular traps (NETs) in diabetic wounds. Mechanistically, LRG1 mediates NETosis in an Akt-dependent manner through TGFß type I receptor kinase ALK5. Taken together, our studies demonstrated that LRG1 derived from bone marrow cells is required for normal wound healing, revealing a physiological role for this glycoprotein, but that excess LRG1 expression in diabetes is pathogenic and contributes to chronic wound formation.

Talin1 controls dendritic cell activation by regulating TLR complex assembly and signaling.

Talin critically controls integrin-dependent cell migration, but its regulatory role in skin dendritic cells (DCs) during inflammatory responses has not been investigated. Here, we show that talin1 regulates not only integrin-dependent Langerhans cell (LC) migration, but also MyD88-dependent Toll-like receptor (TLR)-stimulated DC activation. Talin1-deficient LCs failed to exit the epidermis, resulting in reduced LC migration to skin-draining lymph nodes (sdLNs) and defective skin tolerance induction, while talin1-deficient dermal DCs unexpectedly accumulated in the dermis despite their actomyosin-dependent migratory capabilities. Furthermore, talin1-deficient DCs exhibited compromised chemotaxis, NFκB activation, and proinflammatory cytokine production. Mechanistically, talin1 was required for the formation of preassembled TLR complexes in DCs at steady state via direct interaction with MyD88 and PIP5K. Local production of PIP2 by PIP5K then recruited TIRAP to the preassembled complexes, which were required for TLR signalosome assembly during DC activation. Thus, talin1 regulates MyD88-dependent TLR signaling pathways in DCs through a novel mechanism with implications for antimicrobial and inflammatory immune responses.

Obesity retunes turnover kinetics of tissue-resident macrophages in fat.

Adipose tissue-resident F4/80hi macrophages (ATMs) are the main leukocyte population found in the visceral adipose tissue (VAT). These macrophages comprise several phenotypically distinct subpopulations that rapidly shift in abundance during obesity-induced tissue remodeling. Here we used a fate-mapping approach in mouse models to determine the developmental origins and the differential turnover kinetics of ATMs in lean and obese adipose tissue. We found that in lean, murine VAT the majority of ATMs express T cell immunoglobulin and mucin domain containing 4 receptor (Tim-4), lack the expression of CCR2 and can be further subdivided based on their expression of MHC class II and CD11c. We showed that both embryonic-derived Tim-4+ MHCIIlow and Tim-4+ MHCII+ ATM subsets are long-lived and only slowly replenished by monocytes over time. Only a minor Tim-4- MHCII+ CD11c+ ATM fraction expresses CCR2 and is short-lived. In response to high-fat induced VAT remodeling, the majority of Tim-4+ MHCIIlow ATMs maintain their fetal identity as they are moderately displaced by monocytes. Conversely, Tim-4+ MHCII+ ATMs are quickly replaced in a CCR2-dependent manner by bone marrow-derived Tim-4- MHCII+ ATMs that have significantly higher turnover rates than those in lean mice. In addition, during high-fat diet, the subpopulation of CD11c+ macrophages invade the VAT with the fastest turnover kinetics of all three ATM subpopulations. Our results suggest that ATM subpopulation frequency is controlled by the VAT microenvironment and that obesity-induced tissue remodeling renders some of the ATM niches accessible and available for rapid monocyte replenishment. Specialized monocyte-derived macrophages, which are rapidly recruited may be contributing to control the excess of adipocyte-released lipids produced during obesity.

Islet macrophages are associated with islet vascular remodeling and compensatory hyperinsulinemia during diabetes.

ß-Cells respond to peripheral insulin resistance by first increasing circulating insulin during diabetes. Islet remodeling supports this compensation, but its drivers remain poorly understood. Infiltrating macrophages have been implicated in late-stage type 2 diabetes, but relatively little is known on islet resident macrophages, especially during compensatory hyperinsulinemia. We hypothesized that islet resident macrophages would contribute to islet vascular remodeling and hyperinsulinemia during diabetes, the failure of which results in a rapid progression to frank diabetes. We used chemical (clodronate), genetics (CD169-diphtheria toxin receptor mice), or antibody-mediated (colony-stimulating factor 1 receptor α) macrophage ablation methods in diabetic (db/db) and diet-induced models of compensatory hyperinsulinemia to investigate the role of macrophages in islet remodeling. We transplanted islets devoid of macrophages into naïve diabetic mice and assessed the impact on islet vascularization. With the use of the above methods, we showed that macrophage depletion significantly and consistently compromised islet remodeling in terms of size, vascular density, and insulin secretion capacity. Depletion of islet macrophages reduced VEGF-A secretion in both human and mouse islets ex vivo, and this functionally translated to delayed revascularization upon transplantation in vivo. We revealed that islet resident macrophages were associated with islet remodeling and increased insulin secretion during diabetes. This suggests utility in harnessing islet macrophages during this phase to promote islet vascularization, remodeling, and insulin secretion.

Targeting Mutated Plus Germline Epitopes Confers Pre-clinical Efficacy of an Instantly Formulated Cancer Nano-Vaccine.

Personalized cancer vaccines hold promises for future cancer therapy. Targeting neoantigens is perceived as more beneficial compared to germline, non-mutated antigens. However, it is a practical challenge to identify and vaccinate patients with neoantigens. Here we asked whether two neoantigens are sufficient, and whether the addition of germline antigens would enhance the therapeutic efficacy. We developed and used a personalized cancer nano-vaccine platform based on virus-like particles loaded with toll-like receptor ligands. We generated three sets of multi-target vaccines (MTV) to immunize against the aggressive B16F10 murine melanoma: one set based on germline epitopes (GL-MTV) identified by immunopeptidomics, another set based on mutated epitopes (Mutated-MTV) predicted by whole exome sequencing and a last set combines both germline and mutated epitopes (Mix-MTV). Our results demonstrate that both germline and mutated epitopes induced protection but the best therapeutic effect was achieved with the combination of both. Our platform is based on Cu-free click chemistry used for peptide-VLP coupling, thus enabling bedside production of a personalized cancer vaccine, ready for clinical translation.

TCR Affinity Biases Th Cell Differentiation by Regulating CD25, Eef1e1, and Gbp2.

Naive CD4+ T lymphocytes differentiate into various Th cell subsets following TCR binding to microbial peptide:MHC class II (p:MHCII) complexes on dendritic cells (DCs). The affinity of the TCR interaction with p:MHCII plays a role in Th differentiation by mechanisms that are not completely understood. We found that low-affinity TCRs biased mouse naive T cells to become T follicular helper (Tfh) cells, whereas higher-affinity TCRs promoted the formation of Th1 or Th17 cells. We explored the basis for this phenomenon by focusing on IL-2R signaling, which is known to promote Th1 and suppress Tfh cell differentiation. SIRP⍺+ DCs produce abundant p:MHCII complexes and consume IL-2, whereas XCR1+ DCs weakly produce p:MHCII but do not consume IL-2. We found no evidence, however, of preferential interactions between Th1 cell-prone, high-affinity T cells and XCR1+ DCs or Tfh cell-prone, low-affinity T cells and SIRP⍺+ DCs postinfection with bacteria expressing the peptide of interest. Rather, high-affinity T cells sustained IL-2R expression longer and expressed two novel Th cell differentiation regulators, Eef1e1 and Gbp2, to a higher level than low-affinity T cells. These results suggest that TCR affinity does not influence Th cell differentiation by biasing T cell interactions with IL-2-consuming DCs, but instead, directly regulates genes in naive T cells that control the differentiation process.

Organ-Specific Fate, Recruitment, and Refilling Dynamics of Tissue-Resident Macrophages during Blood-Stage Malaria.

Inflammation-induced disappearance of tissue-resident macrophages represents a key pathogen defense mechanism. Using a model of systemic blood-stage malaria, we studied the dynamics of tissue-resident macrophages in multiple organs to determine how they are depleted and refilled during the course of disease. We show that Plasmodium infection results in a transient loss of embryonically established resident macrophages prior to the parasitemia peak. Fate-mapping analysis reveals that inflammatory monocytes contribute to the repopulation of the emptied niches of splenic red pulp macrophages and hepatic Kupffer cells, while lung alveolar macrophages refill their niche predominantly through self-renewal. Interestingly, the local microenvironment of the spleen and liver can "imprint" the molecular characteristics of fetal-derived macrophages on newly differentiated bone marrow-derived immigrants with remarkably similar gene expression profiles and turnover kinetics. Thus, the mononuclear phagocytic system has developed distinct but effective tissue-specific strategies to replenish emptied niches to guarantee the functional integrity of the system.

DTR-mediated conditional cell ablation-Progress and challenges.

Cell ablation is a valuable complement to mutagenesis for experimentally defining specific cell functions in physiology and pathophysiology in small animal models. One of the most popular ablation strategies involves transgenic expression of a primate diphtheria toxin receptor (DTR) on murine cells that are otherwise resistant to the bacterial exotoxin. The efforts of many laboratories using the DTR approach over the years have yielded numerous valuable insights into specific cell functions. Here, we will discuss the technical aspects of the DTR approach, including the strengths, pitfalls, and future strategies to overcome the shortcomings, highlighting a recent paper published in the European Journal of Immunology [El Hachem et al. Eur. J. Immunol. 2018 https://doi.org/10.1002/eji.201747351]. A particular focus will be given to the application of DTR approach to decipher in vivo functions of the murine myeloid cell compartment.

Clec9A+ Dendritic Cells Are Not Essential for Antitumor CD8+ T Cell Responses Induced by Poly I:C Immunotherapy.

In the steady state, tumors harbor several populations of dendritic cells (DCs) and myeloid cells that are key regulators of the intratumoral immune environment. Among these cells, migratory CD103+ cross-presenting DCs are thought to be critical for tumor-specific CTL responses and tumor resistance. However, it is unclear whether this prominent role also extends to immunotherapy. We used a murine orthotopic mammary tumor model, as well as Clec9A-diphtheria toxin receptor mice that can be depleted of the specialized cross-presenting CD8α+ and CD103+ DC1 subsets, to investigate the role of these DCs in immunotherapy. Treatment with monosodium urate crystals and mycobacteria at the tumor site delayed tumor growth and required DC1s for efficacy. In contrast, treatment with poly I:C was equally effective regardless of DC1 depletion. Neither treatment affected myeloid-derived suppressor cell numbers in the spleen or tumor. Similar experiments using subcutaneous B16 melanoma tumors in BATF3-knockout mice confirmed that CD103+ DCs were not necessary for successful poly I:C immunotherapy. Nevertheless, adaptive immune responses were essential for the response to poly I:C, because mice depleted of CD8+ T cells or all DC subsets were unable to delay tumor growth. In vivo experiments showed that DC1 and DC2 subsets were able to take up tumor Ags, with DC2s making up the larger proportion of lymph node DCs carrying tumor material. Both DC subsets were able to cross-present OVA to OT-I T cells in vitro. Thus, immunotherapy with poly I:C enables multiple DC subsets to cross-present tumor Ag for effective antitumor immune responses.