Kathryn Miller‐Jensen

<p>Supplementary Figure 1</p>

<p>Supplementary Figure 5</p>

<p>Supplementary Figure 4</p>

<p>Supplementary Figure 3</p>

<p>Supplementary Figure 2</p>

This deposit contains the cell–cell communication networks and differential-abundance outputs supporting Bridges et al., "Mapping intratumoral myeloid–T cell communication at single-cell resolution." The networks were generated from a scRNA-seq dataset of CD45+ tumor-infiltrating immune cells in the YUMMER1.7 melanoma model, profiled across five treatment conditions (Control, anti-CD40 agonist, low-dose ICB, high-dose ICB, low-dose ICB + anti-CD40 agonist) and a d8/d10 time course of the combination therapy. A public MC38 dataset (GSE224400) is included as a cross-tumor comparator. Cell–cell interactions were inferred at single-cell resolution with NICHES (Raredon et al., 2023), and differential abundance of communication neighbourhoods between treatment conditions was tested with Milo (Dann et al., 2021) using replicate-level pseudo-bulks. The files released here are the AnnData (.h5ad) outputs that the analysis scripts in the companion GitHub repository load directly to produce all CCC-related figures in the manuscript. Raw 10x Genomics counts (the input to the NICHES pipeline) are deposited separately at NCBI GEO under accession GSE280374. All analysis code is at https://github.com/miller-jensen-lab/Bridgesetal-CCC and a paths-only configuration file (`config_paths.py`) maps each filename below to the figure(s) it produces.

This deposit contains the cell–cell communication networks and differential-abundance outputs supporting Bridges et al., "Mapping intratumoral myeloid–T cell communication at single-cell resolution." The networks were generated from a scRNA-seq dataset of CD45+ tumor-infiltrating immune cells in the YUMMER1.7 melanoma model, profiled across five treatment conditions (Control, anti-CD40 agonist, low-dose ICB, high-dose ICB, low-dose ICB + anti-CD40 agonist) and a d8/d10 time course of the combination therapy. A public MC38 dataset (GSE224400) is included as a cross-tumor comparator. Cell–cell interactions were inferred at single-cell resolution with NICHES (Raredon et al., 2023), and differential abundance of communication neighbourhoods between treatment conditions was tested with Milo (Dann et al., 2021) using replicate-level pseudo-bulks. The files released here are the AnnData (.h5ad) outputs that the analysis scripts in the companion GitHub repository load directly to produce all CCC-related figures in the manuscript. Raw 10x Genomics counts (the input to the NICHES pipeline) are deposited separately at NCBI GEO under accession GSE280374. All analysis code is at https://github.com/miller-jensen-lab/Bridgesetal-CCC and a paths-only configuration file (`config_paths.py`) maps each filename below to the figure(s) it produces.

<p>Supplementary Table S2</p>

<p>Figures S1-9</p>

<p>Supplementary Table S1</p>

<p>Supplementary Table S3</p>

Abstract Cytokine dysregulation during microbial infections can dangerously increase the severity of associated diseases. Non-additive responses to simultaneous activation of toll-like receptor (TLR) signaling pathways are one potential contributor. Here we explored mechanisms underlying the TLR2/MyD88 and TLR3/TRIF-mediated responses in macrophages for canonical pro-inflammatory and antiviral targets including TNF, IL-6, IL-12p40, IFN-β and CXCL10. We found that all targets exhibited characteristic levels of synergistic cytokine activation that varied with TLR-ligand concentration and exposure time. These trends were conserved when the TLR3-ligand polyI:C (PIC) was combined with either of the TLR2-mediated pathogens S. aureus or L. pneumophila . Using pharmacological inhibitors, genetic knockouts, and recombinant cytokines, we explored how TLR2-TLR3-induced paracrine signaling via TNF and IFN-β affected synergy. While TNF synergy is paracrine-independent, IFN-β contributes to synergistic activation of IL-6 and IL-12p40. When IFN-β was directly combined with P3C or S. aureus infection, low concentrations produced modest synergy for both, while high concentrations increased IL-6 but antagonized IL-12p40. Thus, multiple mechanisms regulate TLR2/TLR3-mediated synergistic cytokine activation in macrophages, including type I interferon modulation of TRIF-independent pathogen stimulation that increases inflammatory signaling. These findings have important implications for therapeutic modification of cytokine-driven inflammation, including cytokine storms and the development of vaccine adjuvants.

Summary Tumor vascularization is critical to survival of cancer cells, but is frequently perturbed leading to disorganized angiogenesis and emergence of alternative means of delivery of oxygen and nutrients, such as vasculogenic mimicry (VM). Understanding of VM and its relationship to endothelial vascularization has been hampered by the lack of comprehensive combination of in vivo clinical data and relevant in vitro models. We address this challenge by analyzing glioblastoma (GBM) tumors and clinically isolated cancer cells. This analysis strongly suggests a key role of macrophage-induced controlled cell death in emergence of VM. The results further point to entosis of cancer cells as a critical intermediate state in this process, enabled by mechano-chemical cell heterogeneity. We find evidence that macrophages can regulate endothelial angiogenesis and VM as two alternative vascularization mechanisms. These results reveal mechanistic underpinnings of VM and pave the way to predictive analysis of tumor progression.

<p>Supplementary Figure 3</p>

<p>Supplementary Figure 1</p>

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<div>Abstract<p>Tumor-associated macrophages (TAM) are a heterogeneous population of myeloid cells that dictate the inflammatory tone of the tumor microenvironment. In this study, we unveiled a mechanism by which scavenger receptor cluster of differentiation 36 (CD36) suppresses TAM inflammatory states. CD36 was upregulated in TAMs and associated with immunosuppressive features, and myeloid-specific deletion of CD36 significantly reduced tumor growth. Moreover, CD36-deficient TAMs acquired inflammatory signatures including elevated type-I IFN (IFNI) production, mirroring the inverse correlation between CD36 and IFNI response observed in patients with cancer. IFNI, especially IFNβ, produced by CD36-deficient TAMs directly induced tumor cell quiescence and delayed tumor growth. Mechanistically, CD36 acted as a natural suppressor of IFNI signaling in macrophages through p38 activation downstream of oxidized lipid signaling. These findings establish CD36 as a critical regulator of TAM function and the tumor inflammatory microenvironment, providing additional rationale for pharmacologic inhibition of CD36 to rejuvenate antitumor immunity.</p>Significance:<p>CD36 in tumor-associated macrophages mediates immunosuppression and can be targeted as a therapeutic avenue for stimulating interferon production and increasing the efficacy of immunotherapy.</p></div>

<p>Supplementary Figure 2</p>

<p>Supplementary Figure 4</p>

Tumor-associated macrophages (TAM) are a heterogeneous population of myeloid cells that dictate the inflammatory tone of the tumor microenvironment. In this study, we unveiled a mechanism by which scavenger receptor cluster of differentiation 36 (CD36) suppresses TAM inflammatory states. CD36 was upregulated in TAMs and associated with immunosuppressive features, and myeloid-specific deletion of CD36 significantly reduced tumor growth. Moreover, CD36-deficient TAMs acquired inflammatory signatures including elevated type-I IFN (IFNI) production, mirroring the inverse correlation between CD36 and IFNI response observed in patients with cancer. IFNI, especially IFNβ, produced by CD36-deficient TAMs directly induced tumor cell quiescence and delayed tumor growth. Mechanistically, CD36 acted as a natural suppressor of IFNI signaling in macrophages through p38 activation downstream of oxidized lipid signaling. These findings establish CD36 as a critical regulator of TAM function and the tumor inflammatory microenvironment, providing additional rationale for pharmacologic inhibition of CD36 to rejuvenate antitumor immunity. Significance: CD36 in tumor-associated macrophages mediates immunosuppression and can be targeted as a therapeutic avenue for stimulating interferon production and increasing the efficacy of immunotherapy.

The cellular metabolism of macrophages depends on tissue niches and can control macrophage inflammatory or resolving phenotypes. Yet, the identity of signals within tissue niches that control macrophage metabolism is not well understood. Here, using single-cell RNA sequencing of macrophages in early mouse wounds, we find that, rather than gene expression of canonical inflammatory or resolving polarization markers, metabolic gene expression defines distinct populations of early wound macrophages. Single-cell secretomics and transcriptomics identify inflammatory and resolving cytokines expressed by early wound macrophages, and we show that these signals drive metabolic inputs and mitochondrial metabolism in an age-dependent manner. We show that aging alters the metabolome of early wound macrophages and rewires their metabolism from mitochondria to glycolysis. We further show that macrophage-derived Chi3l3 and IGF-1 can induce metabolic inputs and mitochondrial mass/metabolism in aged and bone marrow-derived macrophages. Together, these findings reveal that macrophage-derived signals drive the mitochondrial metabolism of macrophages within early wounds in an age-dependent manner and have implications for inflammatory diseases, chronic injuries, and age-related inflammatory diseases. In Brief: wound niche and drive mitochondrial-based metabolism. Aging significantly alters macrophage heterogeneity and increases glycolytic metabolism, which can be restored to OxPHOS-based metabolism with young niche cytokines. These findings highlight the importance of the tissue niche in driving macrophage phenotypes, with implications for aging-related impairments in wound healing. Highlights: Single cell transcriptional analysis reveals that reveals that metabolic gene expression identifies distinct macrophage populations in early skin wounds.Single-cell secretomic data show that young macrophages contribute to the wound bed niche by secreting molecules such as IGF-1 and Chi3l3.Old wound macrophages display altered metabolomics, elevated glycolytic metabolism and glucose uptake, and reduced lipid uptake and mitochondrial mass/metabolism.Chi3l3 but not IGF-1 secretion is altered in macrophages in an age dependent manner.Chi3l3 can restore mitochondrial mass/metabolism in aged macrophages.

Effective cancer immunotherapies restore anti-tumor immunity by rewiring cell-cell communication. Treatment-induced changes in communication can be inferred from single-cell RNA-sequencing (scRNA-seq) data, but current methods do not effectively manage heterogeneity within cell types. Here we developed a computational approach to efficiently analyze scRNA-seq-derived, single-cell-resolved cell-cell interactomes, which we applied to determine how agonistic CD40 (CD40ag) alters immune cell crosstalk alone, across tumor models, and in combination with immune checkpoint blockade (ICB). Our analyses suggested that CD40ag improves responses to ICB by targeting both immuno-stimulatory and immunosuppressive macrophage subsets communicating with T cells, and we experimentally validated a spatial basis for these subsets with immunofluorescence and spatial transcriptomics. Moreover, treatment with CD40ag and ICB established coordinated myeloid-T cell interaction hubs that are critical for reestablishing antitumor immunity. Our work advances the biological significance of hypotheses generated from scRNA-seq-derived cell-cell interactomes and supports the clinical translation of myeloid-targeted therapies for ICB-resistant tumors.

<p>Supplementary Table S3</p>

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<p>Supplementary Table S2</p>

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<p>Supplementary Table S2</p>

<div>Abstract<p>Checkpoint inhibitors have revolutionized cancer treatment, but resistance remains a significant clinical challenge. Myeloid cells within the tumor microenvironment can modulate checkpoint resistance by either supporting or suppressing adaptive immune responses. Using an anti-PD-1-resistant mouse melanoma model, we show that targeting the myeloid compartment via CD40 activation and CSF1R blockade in combination with anti-PD-1 results in complete tumor regression in a majority of mice. This triple therapy combination was primarily CD40 agonist-driven in the first 24 hours post-therapy and showed a similar systemic cytokine profile in human patients as was seen in mice. Functional single-cell cytokine secretion profiling of dendritic cells (DCs) using a novel microwell assay identified a CCL22+CCL5+ IL12-secreting DC subset as important early-stage effectors of triple therapy. CD4+ and CD8+ T cells are both critical effectors of treatment, and systems analysis of single-cell RNA-sequencing data supported a role for DC-secreted IL12 in priming T-cell activation and recruitment. Finally, we showed that treatment with a novel IL12 mRNA therapeutic alone was sufficient to overcome PD-1 resistance and cause tumor regression. Overall, we conclude that combining myeloid-based innate immune activation and enhancement of adaptive immunity is a viable strategy to overcome anti-PD-1 resistance.</p></div>

<p>Supplementary Table S3</p>

<p>Figures S1-9</p>

Macrophages and dendritic cells (DCs), although ontogenetically distinct, have overlapping functions and exhibit substantial cell-to-cell heterogeneity that can complicate their identification and obscure innate immune function. In this study, we report that M-CSF-differentiated murine bone marrow-derived macrophages (BMDMs) exhibit extreme heterogeneity in the production of IL-12, a key proinflammatory cytokine linking innate and adaptive immunity. A microwell secretion assay revealed that a small fraction of BMDMs stimulated with LPS secrete most IL-12p40, and we confirmed that this is due to extremely high expression of Il12b, the gene encoding IL-12p40, in a subset of cells. Using an Il12b-YFP reporter mouse, we isolated cells with high LPS-induced Il12b expression and found that this subset was enriched for genes associated with the DC lineage. Single-cell RNA sequencing data confirmed a DC-like subset that differentiates within BMDM cultures that is transcriptionally distinct but could not be isolated by surface marker expression. Although not readily apparent in the resting state, upon LPS stimulation, this subset exhibited a typical DC-associated activation program that is distinct from LPS-induced stochastic BMDM cell-to-cell heterogeneity. Overall, our findings underscore the difficulty in distinguishing macrophages and DCs even in widely used in vitro murine BMDM cultures and could affect the interpretation of some studies that use BMDMs to explore acute inflammatory responses.

Apoptosis and clearance of apoptotic cells via efferocytosis are evolutionarily conserved processes that drive tissue repair. However, the mechanisms by which recognition and clearance of apoptotic cells regulate repair are not fully understood. Here, we use single-cell RNA sequencing to provide a map of the cellular dynamics during early inflammation in mouse skin wounds. We find that apoptotic pathways and efferocytosis receptors are elevated in fibroblasts and immune cells, including resident Lyve1 + macrophages, during inflammation. Interestingly, human diabetic foot wounds upregulate mRNAs for efferocytosis pathway genes and display altered efferocytosis signaling via the receptor Axl and its ligand Gas6 . During early inflammation in mouse wounds, we detect upregulation of Axl in dendritic cells and fibroblasts via TLR3-independent mechanisms. Inhibition studies in vivo in mice reveal that Axl signaling is required for wound repair but is dispensable for efferocytosis. By contrast, inhibition of another efferocytosis receptor, Timd4, in mouse wounds decreases efferocytosis and abrogates wound repair. These data highlight the distinct mechanisms by which apoptotic cell detection coordinates tissue repair and provides potential therapeutic targets for chronic wounds in diabetic patients.

BACKGROUND: Stimulating inflammatory tumor associated macrophages can overcome resistance to PD-(L)1 blockade. We previously conducted a phase I trial of cabiralizumab (anti-CSF1R), sotigalimab (CD40-agonist) and nivolumab. Our current purpose was to study the activity and cellular effects of this three-drug regimen in anti-PD-1-resistant melanoma. METHODS: We employed a Simon's two-stage design and analyzed circulating immune cells from patients treated with this regimen for treatment-related changes. We assessed various dose levels of anti-CSF1R in murine melanoma models and studied the cellular and molecular effects. RESULTS: Thirteen patients were enrolled in the first stage. We observed one (7.7%) confirmed and one (7.7%) unconfirmed partial response, 5 patients had stable disease (38.5%) and 6 disease progression (42.6%). We elected not to proceed to the second stage. CyTOF analysis revealed a reduction in non-classical monocytes. Patients with prolonged stable disease or partial response who remained on study for longer had increased markers of antigen presentation after treatment compared to patients whose disease progressed rapidly. In a murine model, higher anti-CSF1R doses resulted in increased tumor growth and worse survival. Using single-cell RNA-sequencing, we identified a suppressive monocyte/macrophage population in murine tumors exposed to higher doses. CONCLUSIONS: Higher anti-CSF1R doses are inferior to lower doses in a preclinical model, inducing a suppressive macrophage population, and potentially explaining the disappointing results observed in patients. While it is impossible to directly infer human doses from murine studies, careful intra-species evaluation can provide important insight. Cabiralizumab dose optimization is necessary for this patient population with limited treatment options. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT03502330.

A binary classification of macrophage activation as inflammatory or resolving does not capture the diversity of macrophage states observed in tissues. However, framing macrophage activation as a continuous spectrum of states over-looks the intracellular and extracellular networks that regulate and coordinate macrophage responses. Here, we suggest that the systems biology concept of network motifs, which incorporate rules of local molecular interactions, is useful for reframing macrophage activation. Because network motifs can be used to regulate distinct biological functions, they offer a simplified unit that can be compared across organismal, tissue, and disease contexts. Moreover, defining macrophage states as combinations of functional modules regulated by network motifs offers a framework to ultimately predict and target macrophage responses arising in complex environments.

Our skin is constantly exposed to potential damage from the outside world, and it is vital that any injuries are repaired quickly and effectively. Diabetes and many other health conditions can hamper wound healing, resulting in chronic wounds that are both painful and at risk of becoming infected, which can lead to serious illness and death of patients. After an injury to the skin, the wound becomes inflamed as immune cells rush to the site of injury to fight off infection and clear the wound of dead cells and debris. Some of these dead cells will have died by a highly controlled process known as apoptosis. These so-called apoptotic cells display signals on their surface that nearby healthy cells recognize. This triggers the healthy cells to eat the apoptotic cells to remove them from the wound. Previous studies have linked changes in cell death and the removal of dead cells to chronic wounds in patients with diabetes, but it remains unclear how removing dead cells from the wound affects healing. Justynski et al. used a genetic technique called single-cell RNA sequencing to study the patterns of gene activity in mouse skin cells shortly after a wound. The experiments found that, as the area around the wound started to become inflamed, the wounded cells produced signals of apoptosis that in turn triggered nearby healthy cells to remove them. Other signals relating to the removal of dead cells were also widespread in the mouse wounds and treating the wounds with drugs that inhibit these signals resulted in multiple defects in the healing process. Further experiments used the same approach to study samples of tissue taken from foot wounds in human patients with or without diabetes. This revealed that several genes involved in the removal of dead cells were more highly expressed in the wounds of diabetic patients than in the wounds of other individuals. These findings indicate that for wounds to heal properly it is crucial for the body to detect and clear apoptotic cells from the wound site. Further studies building on this work may help to explain why some diabetic patients suffer from chronic wounds and help to develop more effective treatments for them.

<div>Abstract<p>Checkpoint inhibitors have revolutionized cancer treatment, but resistance remains a significant clinical challenge. Myeloid cells within the tumor microenvironment can modulate checkpoint resistance by either supporting or suppressing adaptive immune responses. Using an anti–PD-1–resistant mouse melanoma model, we show that targeting the myeloid compartment via CD40 activation and CSF1R blockade in combination with anti–PD-1 results in complete tumor regression in a majority of mice. This triple therapy combination was primarily CD40 agonist-driven in the first 24 hours after therapy and showed a similar systemic cytokine profile in human patients as was seen in mice. Functional single-cell cytokine secretion profiling of dendritic cells (DC) using a novel microwell assay identified a CCL22<sup>+</sup>CCL5<sup>+</sup> IL12-secreting DC subset as important early-stage effectors of triple therapy. CD4<sup>+</sup> and CD8<sup>+</sup> T cells are both critical effectors of treatment, and systems analysis of single-cell RNA sequencing data supported a role for DC-secreted IL12 in priming T-cell activation and recruitment. Finally, we showed that treatment with a novel IL12 mRNA therapeutic alone was sufficient to overcome PD-1 resistance and cause tumor regression. Overall, we conclude that combining myeloid-based innate immune activation and enhancement of adaptive immunity is a viable strategy to overcome anti–PD-1 resistance.</p></div>

<p>Supplementary Table S1</p>