Modeling T cell temporal response to cancer immunotherapy rationalizes development of combinatorial treatment protocols.

Successful immunotherapy relies on triggering complex responses involving T cell dynamics in tumors and the periphery. Characterizing these responses remains challenging using static human single-cell atlases or mouse models. To address this, we developed a framework for in vivo tracking of tumor-specific CD8+ T cells over time and at single-cell resolution. Our tools facilitate the modeling of gene program dynamics in the tumor microenvironment (TME) and the tumor-draining lymph node (tdLN). Using this approach, we characterize two modes of anti-programmed cell death protein 1 (PD-1) activity, decoupling induced differentiation of tumor-specific activated precursor cells from conventional type 1 dendritic cell (cDC1)-dependent proliferation and recruitment to the TME. We demonstrate that combining anti-PD-1 therapy with anti-4-1BB agonist enhances the recruitment and proliferation of activated precursors, resulting in tumor control. These data suggest that effective response to anti-PD-1 therapy is dependent on sufficient influx of activated precursor CD8+ cells to the TME and highlight the importance of understanding system-level dynamics in optimizing immunotherapies.

Bispecific dendritic-T cell engager potentiates anti-tumor immunity.

Immune checkpoint inhibition treatment using aPD-1 monoclonal antibodies is a promising cancer immunotherapy approach. However, its effect on tumor immunity is narrow, as most patients do not respond to the treatment or suffer from recurrence. We show that the crosstalk between conventional type I dendritic cells (cDC1) and T cells is essential for an effective aPD-1-mediated anti-tumor response. Accordingly, we developed a bispecific DC-T cell engager (BiCE), a reagent that facilitates physical interactions between PD-1+ T cells and cDC1. BiCE treatment promotes the formation of active dendritic/T cell crosstalk in the tumor and tumor-draining lymph nodes. In vivo, single-cell and physical interacting cell analysis demonstrates the distinct and superior immune reprogramming of the tumors and tumor-draining lymph nodes treated with BiCE as compared to conventional aPD-1 treatment. By bridging immune cells, BiCE potentiates cell circuits and communication pathways needed for effective anti-tumor immunity.

Geroprotective interventions converge on gene expression programs of reduced inflammation and restored fatty acid metabolism.

Understanding the mechanisms of geroprotective interventions is central to aging research. We compare four prominent interventions: senolysis, caloric restriction, in vivo partial reprogramming, and heterochronic parabiosis. Using published mice transcriptomic data, we juxtapose these interventions against normal aging. We find a gene expression program common to all four interventions, in which inflammation is reduced and several metabolic processes, especially fatty acid metabolism, are increased. Normal aging exhibits the inverse of this signature across multiple organs and tissues. A similar inverse signature arises in three chronic inflammation disease models in a non-aging context, suggesting that the shift in metabolism occurs downstream of inflammation. Chronic inflammation is also shown to accelerate transcriptomic age. We conclude that a core mechanism of geroprotective interventions acts through the reduction of inflammation with downstream effects that restore fatty acid metabolism. This supports the notion of directly targeting genes associated with these pathways to mitigate age-related deterioration.

Regulation of lymphoid-myeloid lineage bias through regnase-1/3-mediated control of Nfkbiz.

ABSTRACT: Regulation of lineage biases in hematopoietic stem and progenitor cells (HSPCs) is pivotal for balanced hematopoietic output. However, little is known about the mechanism behind lineage choice in HSPCs. Here, we show that messenger RNA (mRNA) decay factors regnase-1 (Reg1; Zc3h12a) and regnase-3 (Reg3; Zc3h12c) are essential for determining lymphoid fate and restricting myeloid differentiation in HSPCs. Loss of Reg1 and Reg3 resulted in severe impairment of lymphopoiesis and a mild increase in myelopoiesis in the bone marrow. Single-cell RNA sequencing analysis revealed that Reg1 and Reg3 regulate lineage directions in HSPCs via the control of a set of myeloid-related genes. Reg1- and Reg3-mediated control of mRNA encoding Nfkbiz, a transcriptional and epigenetic regulator, was essential for balancing lymphoid/myeloid lineage output in HSPCs in vivo. Furthermore, single-cell assay for transposase-accessible chromatin sequencing analysis revealed that Reg1 and Reg3 control the epigenetic landscape on myeloid-related gene loci in early stage HSPCs via Nfkbiz. Consistently, an antisense oligonucleotide designed to inhibit Reg1- and Reg3-mediated Nfkbiz mRNA degradation primed hematopoietic stem cells toward myeloid lineages by enhancing Nfkbiz expression. Collectively, the collaboration between posttranscriptional control and chromatin remodeling by the Reg1/Reg3-Nfkbiz axis governs HSPC lineage biases, ultimately dictating the fate of lymphoid vs myeloid differentiation.

Time-resolved single-cell transcriptomics defines immune trajectories in glioblastoma.

Deciphering the cell-state transitions underlying immune adaptation across time is fundamental for advancing biology. Empirical in vivo genomic technologies that capture cellular dynamics are currently lacking. We present Zman-seq, a single-cell technology recording transcriptomic dynamics across time by introducing time stamps into circulating immune cells, tracking them in tissues for days. Applying Zman-seq resolved cell-state and molecular trajectories of the dysfunctional immune microenvironment in glioblastoma. Within 24 hours of tumor infiltration, cytotoxic natural killer cells transitioned to a dysfunctional program regulated by TGFB1 signaling. Infiltrating monocytes differentiated into immunosuppressive macrophages, characterized by the upregulation of suppressive myeloid checkpoints Trem2, Il18bp, and Arg1, over 36 to 48 hours. Treatment with an antagonistic anti-TREM2 antibody reshaped the tumor microenvironment by redirecting the monocyte trajectory toward pro-inflammatory macrophages. Zman-seq is a broadly applicable technology, enabling empirical measurements of differentiation trajectories, which can enhance the development of more efficacious immunotherapies.

Cholesterol 24-hydroxylase at the choroid plexus contributes to brain immune homeostasis.

The choroid plexus (CP) plays a key role in remotely controlling brain function in health, aging, and disease. Here, we report that CP epithelial cells express the brain-specific cholesterol 24-hydroxylase (CYP46A1) and that its levels are decreased under different mouse and human brain conditions, including amyloidosis, aging, and SARS-CoV-2 infection. Using primary mouse CP cell cultures, we demonstrate that the enzymatic product of CYP46A1, 24(S)-hydroxycholesterol, downregulates inflammatory transcriptomic signatures within the CP, found here to be elevated across multiple neurological conditions. In vitro, the pro-inflammatory cytokine tumor necrosis factor α (TNF-α) downregulates CYP46A1 expression, while overexpression of CYP46A1 or its pharmacological activation in mouse CP organ cultures increases resilience to TNF-α. In vivo, overexpression of CYP46A1 in the CP in transgenic mice with amyloidosis is associated with better cognitive performance and decreased brain inflammation. Our findings suggest that CYP46A1 expression in the CP impacts the role of this niche as a guardian of brain immune homeostasis.

Spatial and Temporal Mapping of Breast Cancer Lung Metastases Identify TREM2 Macrophages as Regulators of the Metastatic Boundary.

Cancer mortality primarily stems from metastatic recurrence, emphasizing the urgent need for developing effective metastasis-targeted immunotherapies. To better understand the cellular and molecular events shaping metastatic niches, we used a spontaneous breast cancer lung metastasis model to create a single-cell atlas spanning different metastatic stages and regions. We found that premetastatic lungs are infiltrated by inflammatory neutrophils and monocytes, followed by the accumulation of suppressive macrophages with the emergence of metastases. Spatial profiling revealed that metastasis-associated immune cells were present in the metastasis core, with the exception of TREM2+ regulatory macrophages uniquely enriched at the metastatic invasive margin, consistent across both murine models and human patient samples. These regulatory macrophages (Mreg) contribute to the formation of an immune-suppressive niche, cloaking tumor cells from immune surveillance. Our study provides a compendium of immune cell dynamics across metastatic stages and niches, informing the development of metastasis-targeting immunotherapies. SIGNIFICANCE: Temporal and spatial single-cell analysis of metastasis stages revealed new players in modulating immune surveillance and suppression. Our study highlights distinct populations of TREM2 macrophages as modulators of the microenvironment in metastasis, and as the key immune determinant defining metastatic niches, pointing to myeloid checkpoints to improve therapeutic strategies. This article is featured in Selected Articles from This Issue, p. 2489.

Neural plate progenitors give rise to both anterior and posterior pituitary cells.

The pituitary is the master neuroendocrine gland, which regulates body homeostasis. It consists of the anterior pituitary/adenohypophysis harboring hormones producing cells and the posterior pituitary/neurohypophysis, which relays the passage of hormones from the brain to the periphery. It is accepted that the adenohypophysis originates from the oral ectoderm (Rathke's pouch), whereas the neural ectoderm contributes to the neurohypophysis. Single-cell transcriptomics of the zebrafish pituitary showed that cyp26b1-positive astroglial pituicytes of the neurohypophysis and prop1-positive adenohypophyseal progenitors expressed common markers implying lineage relatedness. Genetic tracing identifies that, in contrast to the prevailing dogma, neural plate precursors of zebrafish (her4.3+) and mouse (Sox1+) contribute to both neurohypophyseal and a subset of adenohypophyseal cells. Pituicyte-derived retinoic-acid-degrading enzyme Cyp26b1 fine-tunes differentiation of prop1+ progenitors into hormone-producing cells. These results challenge the notion that adenohypophyseal cells are exclusively derived from non-neural ectoderm and demonstrate that crosstalk between neuro- and adeno-hypophyseal cells affects differentiation of pituitary cells.

An immune cell atlas reveals the dynamics of human macrophage specification during prenatal development.

Macrophages are heterogeneous and play critical roles in development and disease, but their diversity, function, and specification remain inadequately understood during human development. We generated a single-cell RNA sequencing map of the dynamics of human macrophage specification from PCW 4-26 across 19 tissues. We identified a microglia-like population and a proangiogenic population in 15 macrophage subtypes. Microglia-like cells, molecularly and morphologically similar to microglia in the CNS, are present in the fetal epidermis, testicle, and heart. They are the major immune population in the early epidermis, exhibit a polarized distribution along the dorsal-lateral-ventral axis, and interact with neural crest cells, modulating their differentiation along the melanocyte lineage. Through spatial and differentiation trajectory analysis, we also showed that proangiogenic macrophages are perivascular across fetal organs and likely yolk-sac-derived as microglia. Our study provides a comprehensive map of the heterogeneity and developmental dynamics of human macrophages and unravels their diverse functions during development.

Single-cell biology: what does the future hold?

In this Editorial, our Chief Editor and members of our Advisory Editorial Board discuss recent breakthroughs, current challenges, and emerging opportunities in single-cell biology and share their vision of "where the field is headed."

Targeted delivery of tumor necrosis factor in combination with CCNU induces a T cell-dependent regression of glioblastoma.

Glioblastoma is the most aggressive primary brain tumor with an unmet need for more effective therapies. Here, we investigated combination therapies based on L19TNF, an antibody-cytokine fusion protein based on tumor necrosis factor that selectively localizes to cancer neovasculature. Using immunocompetent orthotopic glioma mouse models, we identified strong anti-glioma activity of L19TNF in combination with the alkylating agent CCNU, which cured the majority of tumor-bearing mice, whereas monotherapies only had limited efficacy. In situ and ex vivo immunophenotypic and molecular profiling in the mouse models revealed that L19TNF and CCNU induced tumor DNA damage and treatment-associated tumor necrosis. In addition, this combination also up-regulated tumor endothelial cell adhesion molecules, promoted the infiltration of immune cells into the tumor, induced immunostimulatory pathways, and decreased immunosuppression pathways. MHC immunopeptidomics demonstrated that L19TNF and CCNU increased antigen presentation on MHC class I molecules. The antitumor activity was T cell dependent and completely abrogated in immunodeficient mouse models. On the basis of these encouraging results, we translated this treatment combination to patients with glioblastoma. The clinical translation is ongoing but already shows objective responses in three of five patients in the first recurrent glioblastoma patient cohort treated with L19TNF in combination with CCNU (NCT04573192).

In Synergy: Optimizing CAR T Development and Personalizing Patient Care Using Single-Cell Technologies.

Chimeric antigen receptor (CAR) T therapies hold immense promise to revolutionize cancer treatment. Nevertheless, key challenges, primarily in solid tumor settings, continue to hinder the application of this technology. Understanding CAR T-cell mechanism of action, in vivo activity, and clinical implications is essential for harnessing its full therapeutic potential. Single-cell genomics and cell engineering tools are becoming increasingly effective for the comprehensive research of complex biological systems. The convergence of these two technologies can accelerate CAR T-cell development. Here, we examine the potential of applying single-cell multiomics for the development of next-generation CAR T-cell therapies. SIGNIFICANCE: Although CAR T-cell therapies have demonstrated remarkable clinical results in treating cancer, their effectiveness in most patients and tumor types remains limited. Single-cell technologies, which are transforming our understanding of molecular biology, provide new opportunities to overcome the challenges of CAR T-cell therapies. Given the potential of CAR T-cell therapy to tip the balance in the fight against cancer, it is important to understand how single-cell multiomic approaches can be leveraged to develop the next generations of more effective and less toxic CAR T-cell products and to provide powerful decision-making tools for clinicians to optimize treatment and improve patient outcomes.

The transcriptional and regulatory identity of erythropoietin producing cells.

Erythropoietin (Epo) is the master regulator of erythropoiesis and oxygen homeostasis. Despite its physiological importance, the molecular and genomic contexts of the cells responsible for renal Epo production remain unclear, limiting more-effective therapies for anemia. Here, we performed single-cell RNA and transposase-accessible chromatin (ATAC) sequencing of an Epo reporter mouse to molecularly identify Epo-producing cells under hypoxic conditions. Our data indicate that a distinct population of kidney stroma, which we term Norn cells, is the major source of endocrine Epo production in mice. We use these datasets to identify the markers, signaling pathways and transcriptional circuits characteristic of Norn cells. Using single-cell RNA sequencing and RNA in situ hybridization in human kidney tissues, we further provide evidence that this cell population is conserved in humans. These preliminary findings open new avenues to functionally dissect EPO gene regulation in health and disease and may serve as groundwork to improve erythropoiesis-stimulating therapies.

Fc glycoengineering of a PD-L1 antibody harnesses Fcγ receptors for increased antitumor efficacy.

FDA-approved anti-PD-L1 monoclonal antibodies (mAbs) bear the IgG1 isotype, whose scaffolds are either wild-type (e.g., avelumab) or Fc-mutated and lacking Fcγ receptor (FcγR) engagement (e.g., atezolizumab). It is unknown whether variation in the ability of the IgG1 Fc region to engage FcγRs renders mAbs with superior therapeutic activity. In this study, we used humanized FcγR mice to study the contribution of FcγR signaling to the antitumor activity of human anti-PD-L1 mAbs and to identify an optimal human IgG scaffold for PD-L1 mAbs. We observed similar antitumor efficacy and comparable tumor immune responses in mice treated with anti-PD-L1 mAbs with wild-type and Fc-mutated IgG scaffolds. However, in vivo antitumor activity of the wild-type anti-PD-L1 mAb avelumab was enhanced by combination treatment with an FcγRIIB-blocking antibody, which was co-administered to overcome the suppressor function of FcγRIIB in the tumor microenvironment (TME). We performed Fc glycoengineering to remove the fucose subunit from the Fc-attached glycan of avelumab to enhance its binding to the activating FcγRIIIA. Treatment with the Fc-afucosylated version of avelumab also enhanced antitumor activity and induced stronger antitumor immune responses compared with the parental IgG. The enhanced effect by afucosylated PD-L1 antibody was dependent on neutrophils and associated with decreased frequencies of PD-L1+ myeloid cells and increased infiltration of T cells in the TME. Our data reveal that the current design of FDA-approved anti-PD-L1 mAbs does not optimally harness FcγR pathways and suggest two strategies to enhance FcγR engagement to optimize anti-PD-L1 immunotherapy.

Immunometabolism at the crossroads of obesity and cancer-a Keystone Symposia report.

Immunometabolism considers the relationship between metabolism and immunity. Typically, researchers focus on either the metabolic pathways within immune cells that affect their function or the impact of immune cells on systemic metabolism. A more holistic approach that considers both these viewpoints is needed. On September 5-8, 2022, experts in the field of immunometabolism met for the Keystone symposium "Immunometabolism at the Crossroads of Obesity and Cancer" to present recent research across the field of immunometabolism, with the setting of obesity and cancer as an ideal example of the complex interplay between metabolism, immunity, and cancer. Speakers highlighted new insights on the metabolic links between tumor cells and immune cells, with a focus on leveraging unique metabolic vulnerabilities of different cell types in the tumor microenvironment as therapeutic targets and demonstrated the effects of diet, the microbiome, and obesity on immune system function and cancer pathogenesis and therapy. Finally, speakers presented new technologies to interrogate the immune system and uncover novel metabolic pathways important for immunity.

Early Infiltration of Innate Immune Cells to the Liver Depletes HNF4α and Promotes Extrahepatic Carcinogenesis.

Multiple studies have identified metabolic changes within the tumor and its microenvironment during carcinogenesis. Yet, the mechanisms by which tumors affect the host metabolism are unclear. We find that systemic inflammation induced by cancer leads to liver infiltration of myeloid cells at early extrahepatic carcinogenesis. The infiltrating immune cells via IL6-pSTAT3 immune-hepatocyte cross-talk cause the depletion of a master metabolic regulator, HNF4α, consequently leading to systemic metabolic changes that promote breast and pancreatic cancer proliferation and a worse outcome. Preserving HNF4α levels maintains liver metabolism and restricts carcinogenesis. Standard liver biochemical tests can identify early metabolic changes and predict patients' outcomes and weight loss. Thus, the tumor induces early metabolic changes in its macroenvironment with diagnostic and potentially therapeutic implications for the host. SIGNIFICANCE: Cancer growth requires a permanent nutrient supply starting from early disease stages. We find that the tumor extends its effect to the host's liver to obtain nutrients and rewires the systemic and tissue-specific metabolism early during carcinogenesis. Preserving liver metabolism restricts tumor growth and improves cancer outcomes. This article is highlighted in the In This Issue feature, p. 1501.

A single-cell census of mouse limb development identifies complex spatiotemporal dynamics of skeleton formation.

Limb development has long served as a model system for coordinated spatial patterning of progenitor cells. Here, we identify a population of naive limb progenitors and show that they differentiate progressively to form the skeleton in a complex, non-consecutive, three-dimensional pattern. Single-cell RNA sequencing of the developing mouse forelimb identified three progenitor states: naive, proximal, and autopodial, as well as Msx1 as a marker for the naive progenitors. In vivo lineage tracing confirmed this role and localized the naive progenitors to the outer margin of the limb, along the anterior-posterior axis. Sequential pulse-chase experiments showed that the progressive transition of Msx1+ naive progenitors into proximal and autopodial progenitors coincides with their differentiation to Sox9+ chondroprogenitors, which occurs along all the forming skeletal segments. Indeed, tracking the spatiotemporal sequence of differentiation showed that the skeleton forms progressively in a complex pattern. These findings suggest an alternative model for limb skeleton development.

Cerebrospinal fluid proteomics indicates immune dysregulation and neuronal dysfunction in antibody associated autoimmune encephalitis.

Autoimmune Encephalitis (AE) spans a group of non-infectious inflammatory conditions of the central nervous system due to an imbalanced immune response. Aiming to elucidate the pathophysiological mechanisms of AE, we applied an unsupervised proteomic approach to analyze the cerebrospinal fluid (CSF) protein profile of AE patients with autoantibodies against N-methyl-d-aspartate receptor (NMDAR) (n = 9), leucine-rich glioma-inactivated protein 1 (LGI1) (n = 9), or glutamate decarboxylase 65 (GAD65) (n = 8) compared to 9 patients with relapsing-remitting multiple sclerosis as inflammatory controls, and 10 patients with somatic symptom disorder as non-inflammatory controls. We found a dysregulation of the complement system, a disbalance between pro-inflammatory and anti-inflammatory proteins on the one hand, and dysregulation of proteins involved in synaptic transmission, synaptogenesis, brain connectivity, and neurodegeneration on the other hand to a different extent in all AE subtypes compared to non-inflammatory controls. Furthermore, elevated levels of several proteases and reduction in protease inhibitors could be detected in all AE subtypes compared to non-inflammatory controls. Moreover, the different AE subtypes showed distinct protein profiles compared to each other and inflammatory controls which may facilitate future identification of disease-specific biomarkers. Overall, CSF proteomics provides insights into the complex pathophysiological mechanisms of AE, including immune dysregulation, neuronal dysfunction, neurodegeneration, and altered protease function.

Neighbor-specific gene expression revealed from physically interacting cells during mouse embryonic development.

Development of multicellular organisms is orchestrated by persistent cell-cell communication between neighboring partners. Direct interaction between different cell types can induce molecular signals that dictate lineage specification and cell fate decisions. Current single-cell RNA-seq technology cannot adequately analyze cell-cell contact-dependent gene expression, mainly due to the loss of spatial information. To overcome this obstacle and resolve cell-cell contact-specific gene expression during embryogenesis, we performed RNA sequencing of physically interacting cells (PIC-seq) and assessed them alongside similar single-cell transcriptomes derived from developing mouse embryos between embryonic day (E) 7.5 and E9.5. Analysis of the PIC-seq data identified gene expression signatures that were dependent on the presence of specific neighboring cell types. Our computational predictions, validated experimentally, demonstrated that neural progenitor (NP) cells upregulate Lhx5 and Nkx2-1 genes, when exclusively interacting with definitive endoderm (DE) cells. Moreover, there was a reciprocal impact on the transcriptome of DE cells, as they tend to upregulate Rax and Gsc when in contact with NP cells. Using individual cell transcriptome data, we formulated a means of computationally predicting the impact of one cell type on the transcriptome of its neighboring cell types. We have further developed a distinctive spatial-t-distributed stochastic neighboring embedding to display the pseudospatial distribution of cells in a 2-dimensional space. In summary, we describe an innovative approach to study contact-specific gene regulation during embryogenesis.