Nonclassical monocytes potentiate anti-tumoral CD8+ T cell responses in the lungs.

CD8+ T cells drive anti-cancer immunity in response to antigen-presenting cells such as dendritic cells and subpopulations of monocytes and macrophages. While CD14+ classical monocytes modulate CD8+ T cell responses, the contributions of CD16+ nonclassical monocytes to this process remain unclear. Herein we explored the role of nonclassical monocytes in CD8+ T cell activation by utilizing E2-deficient (E2-/-) mice that lack nonclassical monocytes. During early metastatic seeding, modeled by B16F10-OVA cancer cells injected into E2-/- mice, we noted lower CD8+ effector memory and effector T cell frequencies within the lungs as well as in lung-draining mediastinal lymph nodes in the E2-/- mice. Analysis of the myeloid compartment revealed that these changes were associated with depletion of MHC-IIloLy6Clo nonclassical monocytes within these tissues, with little change in other monocyte or macrophage populations. Additionally, nonclassical monocytes preferentially trafficked to primary tumor sites in the lungs, rather than to the lung-draining lymph nodes, and did not cross-present antigen to CD8+ T cells. Examination of the lung microenvironment in E2-/- mice revealed reduced CCL21 expression in endothelial cells, which is chemokine involved in T cell trafficking. Our results highlight the previously unappreciated importance of nonclassical monocytes in shaping the tumor microenvironment via CCL21 production and CD8+ T cell recruitment.

Single-cell immune profiling reveals long-term changes in myeloid cells and identifies a novel subset of CD9+ monocytes associated with COVID-19 hospitalization.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can result in severe immune dysfunction, hospitalization, and death. Many patients also develop long-COVID-19, experiencing symptoms months after infection. Although significant progress has been made in understanding the immune response to acute SARS-CoV-2 infection, gaps remain in our knowledge of how innate immunity influences disease kinetics and severity. We hypothesized that cytometry by time-of-flight analysis of PBMCs from healthy and infected subjects would identify novel cell surface markers and innate immune cell subsets associated with COVID-19 severity. In this pursuit, we identified monocyte and dendritic cell subsets that changed in frequency during acute SARS-CoV-2 infection and correlated with clinical parameters of disease severity. Subsets of nonclassical monocytes decreased in frequency in hospitalized subjects, yet increased in the most severe patients and positively correlated with clinical values associated with worse disease severity. CD9, CD163, PDL1, and PDL2 expression significantly increased in hospitalized subjects, and CD9 and 6-Sulfo LacNac emerged as the markers that best distinguished monocyte subsets amongst all subjects. CD9+ monocytes remained elevated, whereas nonclassical monocytes remained decreased, in the blood of hospitalized subjects at 3-4 months postinfection. Finally, we found that CD9+ monocytes functionally released more IL-8 and MCP-1 after LPS stimulation. This study identifies new monocyte subsets present in the blood of COVID-19 patients that correlate with disease severity, and links CD9+ monocytes to COVID-19 progression.

Analyzing immune response to engineered hydrogels by hierarchical clustering of inflammatory cell subsets.

Understanding the immune response to hydrogel implantation is critical for the design of immunomodulatory biomaterials. To study the progression of inflammation around poly(ethylene glycol) hydrogels presenting Arg-Gly-Asp (RGD) peptides and vascular endothelial growth factor, we used temporal analysis of high-dimensional flow cytometry data paired with intravital imaging, immunohistochemistry, and multiplexed proteomic profiling. RGD-presenting hydrogels created a reparative microenvironment promoting CD206+ cellular infiltration and revascularization in wounded dorsal skin tissue. Unbiased clustering algorithms (SPADE) revealed significant phenotypic transition shifts as a function of the cell-adhesion hydrogel properties. SPADE identified an intermediate macrophage subset functionally regulating in vivo cytokine secretion that was preferentially recruited for RGD-presenting hydrogels, whereas dendritic cell subsets were preferentially recruited to RDG-presenting hydrogels. Last, RGD-presenting hydrogels controlled macrophage functional cytokine secretion to direct polarization and vascularization. Our studies show that unbiased clustering of single-cell data provides unbiased insights into the underlying immune response to engineered materials.

Integrated single-cell transcriptome analysis reveals heterogeneity of esophageal squamous cell carcinoma microenvironment.

The tumor microenvironment is a highly complex ecosystem of diverse cell types, which shape cancer biology and impact the responsiveness to therapy. Here, we analyze the microenvironment of esophageal squamous cell carcinoma (ESCC) using single-cell transcriptome sequencing in 62,161 cells from blood, adjacent nonmalignant and matched tumor samples from 11 ESCC patients. We uncover heterogeneity in most cell types of the ESCC stroma, particularly in the fibroblast and immune cell compartments. We identify a tumor-specific subset of CST1+ myofibroblasts with prognostic values and potential biological significance. CST1+ myofibroblasts are also highly tumor-specific in other cancer types. Additionally, a subset of antigen-presenting fibroblasts is revealed and validated. Analyses of myeloid and T lymphoid lineages highlight the immunosuppressive nature of the ESCC microenvironment, and identify cancer-specific expression of immune checkpoint inhibitors. This work establishes a rich resource of stromal cell types of the ESCC microenvironment for further understanding of ESCC biology.

Nanofiber-Based Delivery of Bioactive Lipids Promotes Pro-regenerative Inflammation and Enhances Muscle Fiber Growth After Volumetric Muscle Loss.

Volumetric muscle loss (VML) injuries after extremity trauma results in an important clinical challenge often associated with impaired healing, significant fibrosis, and long-term pain and functional deficits. While acute muscle injuries typically display a remarkable capacity for regeneration, critically sized VML defects present a dysregulated immune microenvironment which overwhelms innate repair mechanisms leading to chronic inflammation and pro-fibrotic signaling. In this series of studies, we developed an immunomodulatory biomaterial therapy to locally modulate the sphingosine-1-phosphate (S1P) signaling axis and resolve the persistent pro-inflammatory injury niche plaguing a critically sized VML defect. Multiparameter pseudo-temporal 2D projections of single cell cytometry data revealed subtle distinctions in the altered dynamics of specific immune subpopulations infiltrating the defect that were critical to muscle regeneration. We show that S1P receptor modulation via nanofiber delivery of Fingolimod (FTY720) was characterized by increased numbers of pro-regenerative immune subsets and coincided with an enriched pool of muscle stem cells (MuSCs) within the injured tissue. This FTY720-induced priming of the local injury milieu resulted in increased myofiber diameter and alignment across the defect space followed by enhanced revascularization and reinnervation of the injured muscle. These findings indicate that localized modulation of S1P receptor signaling via nanofiber scaffolds, which resemble the native extracellular matrix ablated upon injury, provides great potential as an immunotherapy for bolstering endogenous mechanisms of regeneration following VML injury.

Single-cell transcriptomics identifies gene expression networks driving differentiation and tumorigenesis in the human fallopian tube.

The human fallopian tube harbors the cell of origin for the majority of high-grade serous "ovarian" cancers (HGSCs), but its cellular composition, particularly the epithelial component, is poorly characterized. We perform single-cell transcriptomic profiling of around 53,000 individual cells from 12 primary fallopian specimens to map their major cell types. We identify 10 epithelial subpopulations with diverse transcriptional programs. Based on transcriptional signatures, we reconstruct a trajectory whereby secretory cells differentiate into ciliated cells via a RUNX3high intermediate. Computational deconvolution of advanced HGSCs identifies the "early secretory" population as a likely precursor state for the majority of HGSCs. Its signature comprises both epithelial and mesenchymal features and is enriched in mesenchymal-type HGSCs (p = 6.7 × 10-27), a group known to have particularly poor prognoses. This cellular and molecular compendium of the human fallopian tube in cancer-free women is expected to advance our understanding of the earliest stages of fallopian epithelial neoplasia.

Modulating local S1P receptor signaling as a regenerative immunotherapy after volumetric muscle loss injury.

Regeneration of skeletal muscle after volumetric injury is thought to be impaired by a dysregulated immune microenvironment that hinders endogenous repair mechanisms. Such defects result in fatty infiltration, tissue scarring, chronic inflammation, and debilitating functional deficits. Here, we evaluated the key cellular processes driving dysregulation in the injury niche through localized modulation of sphingosine-1-phosphate (S1P) receptor signaling. We employ dimensionality reduction and pseudotime analysis on single cell cytometry data to reveal heterogeneous immune cell subsets infiltrating preclinical muscle defects due to S1P receptor inhibition. We show that global knockout of S1P receptor 3 (S1PR3) is marked by an increase of muscle stem cells within injured tissue, a reduction in classically activated relative to alternatively activated macrophages, and increased bridging of regenerating myofibers across the defect. We found that local S1PR3 antagonism via nanofiber delivery of VPC01091 replicated key features of pseudotime immune cell recruitment dynamics and enhanced regeneration characteristic of global S1PR3 knockout. Our results indicate that local S1P receptor modulation may provide an effective immunotherapy for promoting a proreparative environment leading to improved regeneration following muscle injury.

Coexpression of CD71 and CD117 Identifies an Early Unipotent Neutrophil Progenitor Population in Human Bone Marrow.

Neutrophils are the most abundant peripheral immune cells and thus, are continually replenished by bone marrow-derived progenitors. Still, how newly identified neutrophil subsets fit into the bone marrow neutrophil lineage remains unclear. Here, we use mass cytometry to show that two recently defined human neutrophil progenitor populations contain a homogeneous progenitor subset we term "early neutrophil progenitors" (eNePs) (Lin-CD66b+CD117+CD71+). Surface marker- and RNA-expression analyses, together with in vitro colony formation and in vivo adoptive humanized mouse transfers, indicate that eNePs are the earliest human neutrophil progenitors. Furthermore, we identified CD71 as a marker associated with the earliest neutrophil developmental stages. Expression of CD71 marks proliferating neutrophils, which were expanded in the blood of melanoma patients and detectable in blood and tumors from lung cancer patients. In summary, we establish CD117+CD71+ eNeP as the inceptive human neutrophil progenitor and propose a refined model of the neutrophil developmental lineage in bone marrow.

Functional crosstalk between T cells and monocytes in cancer and atherosclerosis.

Monocytes and monocyte-derived cells, including Mϕs and dendritic cells, exhibit a diverse array of phenotypic states that are dictated by their surrounding microenvironment. These cells direct T cell activation and function via cues that range from being immunosuppressive to immunostimulatory. Solid tumors and atherosclerotic plaques represent two pathological niches with distinct immune microenvironments. While monocytes and their progeny possess a phenotypic spectrum found within both disease contexts, most within tumors are pro-tumoral and support evasion of host immune responses by tumor cells. In contrast, monocyte-derived cells within atherosclerotic plaques are usually pro-atherogenic, pro-inflammatory, and predominantly directed against self-antigens. Consequently, cancer immunotherapies strive to enhance the immune response against tumor antigens, whereas atherosclerosis treatments seek to dampen the immune response against lipid antigens. Insights into monocyte-T cell interactions within these niches could thus inform therapeutic strategies for two immunologically distinct diseases. Here, we review monocyte diversity, interactions between monocytes and T cells within tumor and plaque microenvironments, how certain therapies have leveraged these interactions, and novel strategies to assay such associations.

Monocyte heterogeneity and functions in cancer.

Monocytes are innate immune cells of the mononuclear phagocyte system that have emerged as important regulators of cancer development and progression. Our understanding of monocytes has advanced from viewing these cells as a homogenous population to a heterogeneous system of cells that display diverse responses to different stimuli. During cancer, different monocyte subsets perform functions that contribute to both pro- and antitumoral immunity, including phagocytosis, secretion of tumoricidal mediators, promotion of angiogenesis, remodeling of the extracellular matrix, recruitment of lymphocytes, and differentiation into tumor-associated macrophages and dendritic cells. The ability of cancer to evade immune recognition and clearance requires protumoral signals to outweigh ongoing attempts by the host immune system to prevent tumor growth. This review discusses current understanding of monocyte heterogeneity during homeostasis, highlights monocyte functions in cancer progression, and describes monocyte-targeted therapeutic strategies for cancer treatment.

Novel Lipid Signaling Mediators for Mesenchymal Stem Cell Mobilization during Bone Repair.

INTRODUCTION: Mesenchymal stem and progenitor cells (MSCs), which normally reside in the bone marrow, are critical to bone health and can be recruited to sites of traumatic bone injury, contributing to new bone formation. The ability to control the trafficking of MSCs provides therapeutic potential for improving traumatic bone healing and therapy for genetic bone diseases such as hypophosphatasia. METHODS: In this study, we explored the sphingosine-1-phosphate (S1P) signaling axis as a means to control the mobilization of MSCs into blood and possibly to recruit MSCs enhancing bone growth. RESULTS: Loss of S1P receptor 3 (S1PR3) leads to an increase in circulating CD45-/CD29+/CD90+/Sca1 putative mesenchymal progenitor cells, suggesting that blocking S1PR3 may stimulate MSCs to leave the bone marrow. Antagonism of S1PR3 with the small molecule VPC01091 stimulated acute migration of CD45-/CD29+/CD90+/Sca1+ MSCs into the blood as early as 1.5 hours after treatment. VPC01091 administration also increased ectopic bone formation induced by BMP-2 and significantly increased new bone formation in critically sized rat cranial defects, suggesting that mobilized MSCs may home to injuries to contribute to healing. We also explored the possibility of combining S1P manipulation of endogenous host cell occupancy with exogenous MSC transplantation for potential use in combination therapies. Importantly, reducing niche occupancy of host MSCs with VPC01091 does not impede engraftment of exogenous MSCs. CONCLUSIONS: Our studies suggest that MSC mobilization through S1PR3 antagonism is a promising strategy for endogenous tissue engineering and improving MSC delivery to treat bone diseases.

An engineered macroencapsulation membrane releasing FTY720 to precondition pancreatic islet transplantation.

Macroencapsulation is a powerful approach to increase the efficiency of extrahepatic pancreatic islet transplant. FTY720, a small molecule that activates signaling through sphingosine-1-phosphate receptors, is immunomodulatory and pro-angiogenic upon sustained delivery from biomaterials. While FTY720 (fingolimod, Gilenya) has been explored for organ transplantation, in the present work the effect of locally released FTY720 from novel nanofiber-based macroencapsulation membranes is explored for islet transplantation. We screened islet viability during culture with FTY720 and various biodegradable polymers. Islet viability is significantly reduced by the addition of high doses (≥500 ng/mL) of soluble FTY720. Among the polymers screened, islets have the highest viability when cultured with poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). Therefore, PHBV was blended with polycaprolactone (PCL) for mechanical stability and electrospun into nanofibers. Islets had no detectable function ex vivo following 5 days or 12 h of subcutaneous implantation within our engineered device. Subsequently, we explored a preconditioning scheme in which islets are transplanted 2 weeks after FTY720-loaded nanofibers are implanted. This allows FTY720 to orchestrate a local regenerative milieu while preventing premature transplantation into avascular sites that contain high concentrations of FTY720. These results provide a foundation and motivation for further investigation into the use of FTY720 in preconditioning sites for efficacious islet transplantation. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 555-568, 2018.

Enrichment of endogenous fractalkine and anti-inflammatory cells via aptamer-functionalized hydrogels.

Early recruitment of non-classical monocytes and their macrophage derivatives is associated with augmented tissue repair and improved integration of biomaterial constructs. A promising therapeutic approach to recruit these subpopulations is by elevating local concentrations of chemoattractants such as fractalkine (FKN, CX3CL1). However, delivering recombinant or purified proteins is not ideal due to their short half-lives, suboptimal efficacy, immunogenic potential, batch variabilities, and cost. Here we report an approach to enrich endogenous FKN, obviating the need for delivery of exogenous proteins. In this study, modified FKN-binding-aptamers are integrated with poly(ethylene glycol) diacrylate to form aptamer-functionalized hydrogels ("aptagels") that localize, dramatically enrich and passively release FKN in vitro for at least one week. Implantation in a mouse model of excisional skin injury demonstrates that aptagels enrich endogenous FKN and stimulate significant local increases in Ly6CloCX3CR1hi non-classical monocytes and CD206+ M2-like macrophages. The results demonstrate that orchestrators of inflammation can be manipulated without delivery of foreign proteins or cells and FKN-aptamer functionalized biomaterials may be a promising approach to recruit anti-inflammatory subpopulations to sites of injury. Aptagels are readily synthesized, highly customizable and could combine different aptamers to treat complex diseases in which regulation or enrichment of multiple proteins may be therapeutic.

Non-classical monocytes are biased progenitors of wound healing macrophages during soft tissue injury.

Successful tissue repair requires the activities of myeloid cells such as monocytes and macrophages that guide the progression of inflammation and healing outcome. Immunoregenerative materials leverage the function of endogenous immune cells to orchestrate complex mechanisms of repair; however, a deeper understanding of innate immune cell function in inflamed tissues and their subsequent interactions with implanted materials is necessary to guide the design of these materials. Blood monocytes exist in two primary subpopulations, characterized as classical inflammatory or non-classical. While classical monocytes extravasate into inflamed tissue and give rise to macrophages or dendritic cells, the recruitment kinetics and functional role of non-classical monocytes remains unclear. Here, we demonstrate that circulating non-classical monocytes are directly recruited to polymer films within skin injuries, where they home to a perivascular niche and generate alternatively activated, wound healing macrophages. Selective labeling of blood monocyte subsets indicates that non-classical monocytes are biased progenitors of alternatively activated macrophages. On-site delivery of the immunomodulatory small molecule FTY720 recruits S1PR3-expressing non-classical monocytes that support vascular remodeling after injury. These results elucidate a previously unknown role for blood-derived non-classical monocytes as contributors to alternatively activated macrophages, highlighting them as key regulators of inflammatory response and regenerative outcome.

Aspirin-Triggered Resolvin D1-modified materials promote the accumulation of pro-regenerative immune cell subsets and enhance vascular remodeling.

Many goals in tissue engineering rely on modulating cellular localization and polarization of cell signaling, including the inhibition of inflammatory infiltrate, facilitation of inflammatory cell egress, and clearance of apoptotic cells. Omega-3 polyunsaturated fatty acid-derived resolvins are gaining increasing recognition for their essential roles in inhibition of neutrophil invasion into inflamed tissue and promotion of macrophage phagocytosis of cellular debris as well as their egress to the lymphatics. Biomaterial-based release of lipid mediators is a largely under-explored approach that provides a method to manipulate local lipid signaling gradients in vivo and direct the recruitment and/or polarization of anti-inflammatory cell subsets to suppress inflammatory signaling and enhance angiogenesis and tissue regeneration. The goal of this study was to encapsulate Aspirin-Triggered Resolvin D1 (AT-RvD1) into a degradable biomaterial in order to elucidate the effects of sustained, localized delivery in a model of sterile inflammation. Flow cytometric and imaging analysis at both 1 and 3days after injury showed that localized AT-RvD1 delivery was able significantly increase the accumulation of anti-inflammatory monocytes and M2 macrophages while limiting the infiltration of neutrophils. Additionally, cytokine profiling and longitudinal vascular analysis revealed a shift towards a pro-angiogenic profile with increased concentrations of VEGF and SDF-1α, and increased arteriolar diameter and tortuosity. These results demonstrate the ability of locally-delivered AT-RvD1 to increase pro-regenerative immune subpopulations and promote vascular remodeling. STATEMENT OF SIGNIFICANCE: This work is motivated by our efforts to explore the underlying mechanisms of inflammation resolution after injury and to develop biomaterial-based approaches to amplify endogenous mechanisms of resolution and repair. Though specific lipid mediators have been identified that actively promote the resolution of inflammation, biomaterial-based localized delivery of these mediators has been largely unexplored. We loaded Aspirin-Triggered Resolvin D1 into a PLGA scaffold and examined the effects of sustained, localized delivery on the innate immune response. We found that biomaterial delivery of resolvin was able to enhance the accumulation of pro-regenerative populations of immune cells, including anti-inflammatory monocytes, population that has never before been shown to respond to resolvin treatment, and also enhance vascular remodeling in response to tissue injury.

Selective recruitment of non-classical monocytes promotes skeletal muscle repair.

Regeneration of traumatic defects in skeletal muscle requires the synchronized behavior of multiple cells that participate in repair. The inflammatory cascade that is rapidly initiated after injury serves as a powerful node at which to guide the progression of healing and influence tissue repair. Here, we examine the role that myeloid cells play in the healing of traumatic skeletal muscle injury, and leverage their pro-regenerative functions using local delivery of the immunomodulatory small molecule FTY720. We demonstrate that increasing the frequency of non-classical monocytes in inflamed muscle coincides with increased numbers of CD206+ alternatively activated macrophages. Animals treated with immunomodulatory materials had greater defect closure and more vascularization in the acute phases of injury. In the later stages of repair, during which parenchymal tissue growth occurs, we observed improved regeneration of muscle fibers and decreased fibrotic tissue following localization of pro-regenerative inflammation. These results highlight non-classical monocytes as a novel therapeutic target to improve the regenerative outcome after traumatic skeletal muscle injury.

Sphingosine-1-Phosphate Receptor-3 Supports Hematopoietic Stem and Progenitor Cell Residence Within the Bone Marrow Niche.

Hematopoietic stem and progenitor cells (HSPCs) egress from bone marrow (BM) during homeostasis and at increased rates during stress; however, the mechanisms regulating their trafficking remain incompletely understood. Here we describe a novel role for lipid receptor, sphingosine-1-phosphate receptor 3 (S1PR3), in HSPC residence within the BM niche. HSPCs expressed increased levels of S1PR3 compared to differentiated BM cells. Pharmacological antagonism or knockout (KO) of S1PR3 mobilized HSPCs into blood circulation, suggesting that S1PR3 influences niche localization. S1PR3 antagonism suppressed BM and plasma SDF-1, enabling HSPCs to migrate toward S1P-rich plasma. Mobilization synergized with AMD3100-mediated antagonism of CXCR4, which tethers HSPCs in the niche, and recovered homing deficits of AMD3100-treated grafts. S1PR3 antagonism combined with AMD3100 improved re-engraftment and survival in lethally irradiated recipients. Our studies indicate that S1PR3 and CXCR4 signaling cooperate to maintain HSPCs within the niche under homeostasis. These results highlight an important role for S1PR3 in HSPC niche occupancy and trafficking that can be harnessed for both rapid clinical stem cell mobilization and re-engraftment strategies, as well as the opportunity to design novel therapeutics for control of recruitment, homing, and localization through bioactive lipid signaling. Stem Cells 2017;35:1040-1052.