RESUMEN
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.
Asunto(s)
Linfocitos T CD8-positivos , Inmunoterapia , Microambiente Tumoral , Animales , Ratones , Inmunoterapia/métodos , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/efectos de los fármacos , Microambiente Tumoral/inmunología , Humanos , Neoplasias/inmunología , Neoplasias/terapia , Neoplasias/tratamiento farmacológico , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Células Dendríticas/inmunología , Células Dendríticas/efectos de los fármacos , Línea Celular TumoralRESUMEN
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.
Asunto(s)
Anemia , Eritropoyetina , Animales , Humanos , Ratones , Anemia/genética , Eritropoyesis/genética , Eritropoyetina/genética , Riñón/metabolismo , ARN/metabolismoRESUMEN
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.
Asunto(s)
Extremidades , Esqueleto , Animales , Ratones , Diferenciación Celular , Extremidades/crecimiento & desarrollo , Organogénesis , Esqueleto/crecimiento & desarrolloRESUMEN
Systemic sclerosis (scleroderma, SSc) is an incurable autoimmune disease with high morbidity and mortality rates. Here, we conducted a population-scale single-cell genomic analysis of skin and blood samples of 56 healthy controls and 97 SSc patients at different stages of the disease. We found immune compartment dysfunction only in a specific subtype of diffuse SSc patients but global dysregulation of the stromal compartment, particularly in a previously undefined subset of LGR5+-scleroderma-associated fibroblasts (ScAFs). ScAFs are perturbed morphologically and molecularly in SSc patients. Single-cell multiome profiling of stromal cells revealed ScAF-specific markers, pathways, regulatory elements, and transcription factors underlining disease development. Systematic analysis of these molecular features with clinical metadata associates specific ScAF targets with disease pathogenesis and SSc clinical traits. Our high-resolution atlas of the sclerodermatous skin spectrum will enable a paradigm shift in the understanding of SSc disease and facilitate the development of biomarkers and therapeutic strategies.
Asunto(s)
Esclerodermia Sistémica , Células Cultivadas , Fibroblastos/metabolismo , Fibrosis , Humanos , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Esclerodermia Sistémica/tratamiento farmacológico , Esclerodermia Sistémica/genética , Piel/metabolismoRESUMEN
Despite their key regulatory role and therapeutic potency, the molecular signatures of interactions between T cells and antigen-presenting myeloid cells within the tumor microenvironment remain poorly characterized. Here, we systematically characterize these interactions using RNA sequencing of physically interacting cells (PIC-seq) and find that CD4+PD-1+CXCL13+ T cells are a major interacting hub with antigen-presenting cells in the tumor microenvironment of human non-small cell lung carcinoma. We define this clonally expanded, tumor-specific and conserved T-cell subset as T-helper tumor (Tht) cells. Reconstitution of Tht cells in vitro and in an ovalbumin-specific αß TCR CD4+ T-cell mouse model, shows that the Tht program is primed in tumor-draining lymph nodes by dendritic cells presenting tumor antigens, and that their function is important for harnessing the antitumor response of anti-PD-1 treatment. Our molecular and functional findings support the modulation of Tht-dendritic cell interaction checkpoints as a major interventional strategy in immunotherapy.
Asunto(s)
Neoplasias Pulmonares , Microambiente Tumoral , Animales , Línea Celular Tumoral , Células Dendríticas , Inhibidores de Puntos de Control Inmunológico/farmacología , Neoplasias Pulmonares/terapia , Ratones , Linfocitos T Colaboradores-InductoresRESUMEN
Multiple myeloma (MM) is a neoplastic plasma-cell disorder characterized by clonal proliferation of malignant plasma cells. Despite extensive research, disease heterogeneity within and between treatment-resistant patients is poorly characterized. In the present study, we conduct a prospective, multicenter, single-arm clinical trial (NCT04065789), combined with longitudinal single-cell RNA-sequencing (scRNA-seq) to study the molecular dynamics of MM resistance mechanisms. Newly diagnosed MM patients (41), who either failed to respond or experienced early relapse after a bortezomib-containing induction regimen, were enrolled to evaluate the safety and efficacy of a daratumumab, carfilzomib, lenalidomide and dexamethasone combination. The primary clinical endpoint was safety and tolerability. Secondary endpoints included overall response rate, progression-free survival and overall survival. Treatment was safe and well tolerated; deep and durable responses were achieved. In prespecified exploratory analyses, comparison of 41 primary refractory and early relapsed patients, with 11 healthy subjects and 15 newly diagnosed MM patients, revealed new MM molecular pathways of resistance, including hypoxia tolerance, protein folding and mitochondria respiration, which generalized to larger clinical cohorts (CoMMpass). We found peptidylprolyl isomerase A (PPIA), a central enzyme in the protein-folding response pathway, as a potential new target for resistant MM. CRISPR-Cas9 deletion of PPIA or inhibition of PPIA with a small molecule inhibitor (ciclosporin) significantly sensitizes MM tumor cells to proteasome inhibitors. Together, our study defines a roadmap for integrating scRNA-seq in clinical trials, identifies a signature of highly resistant MM patients and discovers PPIA as a potent therapeutic target for these tumors.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/patología , Análisis de la Célula Individual/métodos , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Monoclonales/administración & dosificación , Estudios de Casos y Controles , Dexametasona/administración & dosificación , Resistencia a Antineoplásicos , Femenino , Humanos , Lenalidomida/administración & dosificación , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia , Oligopéptidos/administración & dosificación , Resultado del TratamientoRESUMEN
Tertiary lymphoid structures (TLS) are organized aggregates of B and T cells formed ectopically during different stages of life in response to inflammation, infection, or cancer. Here, we describe formation of structures reminiscent of TLS in the spinal cord meninges under several central nervous system (CNS) pathologies. After acute spinal cord injury, B and T lymphocytes locally aggregate within the meninges to form TLS-like structures, and continue to accumulate during the late phase of the response to the injury, with a negative impact on subsequent pathological conditions, such as experimental autoimmune encephalomyelitis. Using a chronic model of spinal cord pathology, the mSOD1 mouse model of amyotrophic lateral sclerosis, we further showed by single-cell RNA-sequencing that a meningeal lymphocyte niche forms, with a unique organization and activation state, including accumulation of pre-B cells in the spinal cord meninges. Such a response was not found in the CNS-draining cervical lymph nodes. The present findings suggest that a special immune response develops in the meninges during various neurological pathologies in the CNS, a possible reflection of its immune privileged nature.
Asunto(s)
Esclerosis Amiotrófica Lateral/inmunología , Linfocitos B/inmunología , Inmunidad , Meninges/inmunología , Traumatismos de la Médula Espinal/inmunología , Linfocitos T/inmunología , Estructuras Linfoides Terciarias/inmunología , Enfermedad Aguda , Animales , Enfermedad Crónica , Modelos Animales de Enfermedad , Inflamación/inmunología , Ganglios Linfáticos/inmunología , Activación de Linfocitos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Cuello , Vértebras Torácicas/lesionesRESUMEN
Crosstalk between neighboring cells underlies many biological processes, including cell signaling, proliferation and differentiation. Current single-cell genomic technologies profile each cell separately after tissue dissociation, losing information on cell-cell interactions. In the present study, we present an approach for sequencing physically interacting cells (PIC-seq), which combines cell sorting of physically interacting cells (PICs) with single-cell RNA-sequencing. Using computational modeling, PIC-seq systematically maps in situ cellular interactions and characterizes their molecular crosstalk. We apply PIC-seq to interrogate diverse interactions including immune-epithelial PICs in neonatal murine lungs. Focusing on interactions between T cells and dendritic cells (DCs) in vitro and in vivo, we map T cell-DC interaction preferences, and discover regulatory T cells as a major T cell subtype interacting with DCs in mouse draining lymph nodes. Analysis of T cell-DC pairs reveals an interaction-specific program between pathogen-presenting migratory DCs and T cells. PIC-seq provides a direct and broadly applicable technology to characterize intercellular interaction-specific pathways at high resolution.
Asunto(s)
Células Dendríticas/citología , Perfilación de la Expresión Génica/métodos , Análisis de la Célula Individual/métodos , Linfocitos T/citología , Algoritmos , Animales , Animales Recién Nacidos , Comunicación Celular , Células Cultivadas , Biología Computacional , Células Dendríticas/química , Femenino , Citometría de Flujo , Pulmón/química , Pulmón/citología , Ratones , Análisis de Secuencia de ARN , Linfocitos T/químicaRESUMEN
Multiple myeloma, a plasma cell malignancy, is the second most common blood cancer. Despite extensive research, disease heterogeneity is poorly characterized, hampering efforts for early diagnosis and improved treatments. Here, we apply single cell RNA sequencing to study the heterogeneity of 40 individuals along the multiple myeloma progression spectrum, including 11 healthy controls, demonstrating high interindividual variability that can be explained by expression of known multiple myeloma drivers and additional putative factors. We identify extensive subclonal structures for 10 of 29 individuals with multiple myeloma. In asymptomatic individuals with early disease and in those with minimal residual disease post-treatment, we detect rare tumor plasma cells with molecular characteristics similar to those of active myeloma, with possible implications for personalized therapies. Single cell analysis of rare circulating tumor cells allows for accurate liquid biopsy and detection of malignant plasma cells, which reflect bone marrow disease. Our work establishes single cell RNA sequencing for dissecting blood malignancies and devising detailed molecular characterization of tumor cells in symptomatic and asymptomatic patients.
Asunto(s)
Heterogeneidad Genética , Mieloma Múltiple/sangre , Neoplasia Residual/sangre , Mieloma Múltiple Quiescente/sangre , Adulto , Anciano , Anciano de 80 o más Años , Secuencia de Bases , Progresión de la Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Mieloma Múltiple/genética , Mieloma Múltiple/patología , Neoplasia Residual/genética , Neoplasia Residual/patología , Mieloma Múltiple Quiescente/genética , Mieloma Múltiple Quiescente/patologíaRESUMEN
Hodgkin lymphoma (HL) is one of the most curable malignancies. Despite its effectiveness, chemotherapy is often associated with adverse events (AEs) such as nausea, anorexia, and impairment of general well-being. Our objective was to assess the extent of medical cannabis use among HL patients and evaluate its efficacy in controlling chemotherapy-related AEs. Patterns of medical cannabis use and efficacy were evaluated using physician-completed application forms, medical files, and patient-completed questionnaires, for all consecutive adult HL patients treated at the Tel-Aviv Medical Center between June 2010 and November 2016. One-hundred and thirty-three patients met the inclusion criteria. The median age of the cohort was 37 years, 53% were male, 46% were diagnosed at an early stage, and 88% achieved a complete response to treatment. Fifty-one patients (38%) used medical cannabis. There were no significant differences in baseline characteristics between cannabis users and nonusers. Cannabis users reported improvement in pain, general well-being, appetite, and nausea in 94, 87, 82, and 79% of cases, respectively. Importantly, 81.5% reported a high overall efficacy of cannabis in relieving symptoms. AEs related to cannabis use itself were mild. Thus, medical cannabis use is prevalent in this HL cohort, and appears to be effective in ameliorating chemotherapy-related AEs.
Asunto(s)
Enfermedad de Hodgkin/tratamiento farmacológico , Marihuana Medicinal/uso terapéutico , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Femenino , Enfermedad de Hodgkin/patología , Humanos , Masculino , Marihuana Medicinal/efectos adversos , Persona de Mediana Edad , Náusea/etiología , Estadificación de Neoplasias , Manejo del Dolor , Pronóstico , Encuestas y Cuestionarios , Resultado del TratamientoRESUMEN
Lung development and function arises from the interactions between diverse cell types and lineages. Using single-cell RNA sequencing (RNA-seq), we characterize the cellular composition of the lung during development and identify vast dynamics in cell composition and their molecular characteristics. Analyzing 818 ligand-receptor interaction pairs within and between cell lineages, we identify broadly interacting cells, including AT2, innate lymphocytes (ILCs), and basophils. Using interleukin (IL)-33 receptor knockout mice and in vitro experiments, we show that basophils establish a lung-specific function imprinted by IL-33 and granulocyte-macrophage colony-stimulating factor (GM-CSF), characterized by unique signaling of cytokines and growth factors important for stromal, epithelial, and myeloid cell fates. Antibody-depletion strategies, diphtheria toxin-mediated selective depletion of basophils, and co-culture studies show that lung resident basophils are important regulators of alveolar macrophage development and function. Together, our study demonstrates how whole-tissue signaling interaction map on the single-cell level can broaden our understanding of cellular networks in health and disease.
Asunto(s)
Basófilos/metabolismo , Comunicación Celular , Impresión Genómica , Macrófagos Alveolares/metabolismo , Transcriptoma , Animales , Diferenciación Celular , Línea Celular Tumoral , Células Cultivadas , Femenino , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Interleucina-33/metabolismo , Macrófagos Alveolares/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Transducción de Señal , Análisis de la Célula IndividualRESUMEN
BACKGROUND: Relatives of patients with chronic lymphocytic leukemia (CLL) are at increased risk of developing CLL. Familial CLL is defined as more than one case of CLL among blood relatives, a phenomenon reported in approximately 5%-10% of all CLL patients. OBJECTIVE: Given the known predisposition of CLL among Ashkenazi Jews, we studied the features of familial CLL in an Israeli population. METHODS: This is a retrospective study, in which we reviewed the demographics, clinical characteristics, and outcomes of a total of 332 patients with CLL/small lymphocytic lymphoma. RESULTS: Familial CLL was recorded in 41 cases (12.3%) of the patients. The age at diagnosis was younger in patients with familial CLL (by almost 3.5 years). Familial CLL was strongly associated with Ashkenazi Jewish origin. Patients with familial CLL more commonly presented with higher hemoglobin and lower serum ß-2-microglobulin levels. No significant differences were detected between sporadic and familial CLL in disease stage, time to treatment, second cancers, or overall survival. CONCLUSION: Familial cases of CLL in an Israeli population show a disproportionate ethnic distribution toward Jews of Ashkenazi origin. The clinical characteristics and the overall outcome are not substantially different from sporadic cases.