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BACKGROUND: While melanomas commonly harbor losses of 9p21, on which CDKN2A resides, the presence of additional tumor suppressor elements at this locus is incompletely characterized. Here we assess the expression levels and functional role of microRNA-876-3p (miR-876), whose gene also maps to 9p21. METHODS: Expression of miR-876 was assessed in human tissues and cell lines using quantitative miRNA reverse transcriptase polymerase chain reaction (qRT-PCR). MIR876 copy number was determined in The Cancer Genome Atlas (TCGA) melanoma cohort. The consequences of regulation of miR-876 expression were assessed on melanoma cell colony formation, migration, invasion, apoptosis, cell cycle progression, and drug sensitivity in culture, and on in vivo tumor growth in a xenograft model. Genome-wide transcriptomic changes induced by miR-876 overexpression were determined using RNA sequencing (RNA-Seq). RESULTS: miR-876 expression was significantly decreased in primary melanoma samples when compared with nevi, and in human melanoma cell lines when compared with human melanocytes. Analysis of the TCGA cohort revealed deletions in MIR876 in > 50% of melanomas. miR-876 overexpression resulted in decreased melanoma cell colony formation, migration, and invasion, which was accompanied by cell cycle arrest and increased apoptosis. Intra-tumoral injections of miR-876 significantly suppressed melanoma growth in vivo. RNA-Seq analysis of miR-876-treated tumors revealed downregulation of several growth-promoting genes, along with upregulation of tumor suppressor genes, which was confirmed by qRT-PCR analysis. Computational analyses identified MAPK1 (or ERK2) as a possible target of miR-876 action. Overexpression of miR-876 significantly suppressed luciferase expression driven by the MAPK1/ERK2 3' UTR, and resulted in decreased ERK protein expression in melanoma cells. MAPK1/ERK2 cDNA overexpression rescued the effects of miR-876 on melanoma colony formation. miR-876 overexpression sensitized melanoma cells to treatment with the BRAF inhibitor vemurafenib. CONCLUSIONS: These studies identify miR-876 as a distinct tumor suppressor on 9p21 that is inactivated in melanoma and suggest miR-876 loss as an additional mechanism to activate ERK and the mitogen activated protein kinase (MAPK) pathway in melanoma. In addition, they suggest the therapeutic potential of combining miR-876 overexpression with BRAF inhibition as a rational therapeutic strategy for melanoma.
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Cromosomas Humanos Par 9 , Regulación Neoplásica de la Expresión Génica , Genes Supresores de Tumor , Melanoma , MicroARNs , Humanos , Melanoma/genética , Melanoma/patología , MicroARNs/genética , MicroARNs/metabolismo , Línea Celular Tumoral , Animales , Cromosomas Humanos Par 9/genética , Movimiento Celular/genética , Apoptosis/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proliferación Celular/genética , Secuencia de Bases , Invasividad NeoplásicaRESUMEN
Fibrosing cholangiopathies, including biliary atresia and primary sclerosing cholangitis, involve immune-mediated bile duct epithelial injury and hepatic bile acid (BA) retention (cholestasis). Regulatory T-cells (Tregs) can prevent auto-reactive lymphocyte activation, yet the effects of BA on this CD4 lymphocyte subset are unknown. Gene regulatory networks for hepatic CD4 lymphocytes in a murine cholestasis model revealed Tregs are polarized to Th17 during cholestasis. Following bile duct ligation, Stat3 deletion in CD4 lymphocytes preserved hepatic Treg responses. While pharmacological reduction of hepatic BA in MDR2-/- mice prompted Treg expansion and diminished liver injury, this improvement subsided with Treg depletion. A cluster of patients diagnosed with biliary atresia showed both increased hepatic Treg responses and improved 2-year native liver survival, supporting that Tregs might protect against neonatal bile duct obstruction. Together, these findings suggest liver BA determine Treg function and should be considered as a therapeutic target to restore protective hepatic immune responses.
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While immunotherapy is typically reserved for cancer patients with a high mutational burden, neoantigens produced from post-transcriptional regulation provide a possible untapped reservoir of common immunogenic targets for new targeted cancer therapies. In this review, we describe new and emerging technologies, unconventional molecular targets and challenges for the precision immune targeting of diverse malignancies. In particular, we focus on the unique potential of targeting alternative mRNA isoforms as a source for broadly presented neoantigens and cell surface proteins. Finally, we discuss emerging challenges for alternative isoform immune targeting, with an emphasis in silico prioritization and high-throughput target validation.
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Inmunoterapia , Neoplasias , Isoformas de ARN , Humanos , Neoplasias/terapia , Neoplasias/inmunología , Neoplasias/genética , Inmunoterapia/métodos , Isoformas de ARN/genética , Antígenos de Neoplasias/inmunología , Antígenos de Neoplasias/genética , AnimalesRESUMEN
Affinity-matured plasma cells (PCs) of varying lifespans are generated through a germinal center (GC) response. The developmental dynamics and genomic programs of antigen-specific PC precursors remain to be elucidated. Here, using a model antigen in mice, we demonstrate biphasic generation of PC precursors, with those generating long-lived bone marrow PCs preferentially produced in the late phase of GC response. Clonal tracing using single-cell RNA sequencing and B cell antigen receptor sequencing in spleen and bone marrow compartments, coupled with adoptive transfer experiments, reveals a new PC transition state that gives rise to functionally competent PC precursors. The latter undergo clonal expansion, dependent on inducible expression of TIGIT. We propose a model for the proliferation and programming of precursors of long-lived PCs, based on extended antigen encounters in the GC.
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Diferenciación Celular , Centro Germinal , Células Plasmáticas , Animales , Células Plasmáticas/inmunología , Células Plasmáticas/metabolismo , Ratones , Centro Germinal/inmunología , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores de Antígenos de Linfocitos B/genética , Ratones Endogámicos C57BL , Receptores Inmunológicos/metabolismo , Receptores Inmunológicos/genética , Ratones TransgénicosRESUMEN
Most human Transcription factors (TFs) genes encode multiple protein isoforms differing in DNA binding domains, effector domains, or other protein regions. The global extent to which this results in functional differences between isoforms remains unknown. Here, we systematically compared 693 isoforms of 246 TF genes, assessing DNA binding, protein binding, transcriptional activation, subcellular localization, and condensate formation. Relative to reference isoforms, two-thirds of alternative TF isoforms exhibit differences in one or more molecular activities, which often could not be predicted from sequence. We observed two primary categories of alternative TF isoforms: "rewirers" and "negative regulators", both of which were associated with differentiation and cancer. Our results support a model wherein the relative expression levels of, and interactions involving, TF isoforms add an understudied layer of complexity to gene regulatory networks, demonstrating the importance of isoform-aware characterization of TF functions and providing a rich resource for further studies.
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Analysis of the human hematopoietic progenitor compartment is being transformed by single-cell multimodal approaches. Cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) enables coupled surface protein and transcriptome profiling, thereby revealing genomic programs underlying progenitor states. To perform CITE-seq systematically on primary human bone marrow cells, we used titrations with 266 CITE-seq antibodies (antibody-derived tags) and machine learning to optimize a panel of 132 antibodies. Multimodal analysis resolved >80 stem, progenitor, immune, stromal and transitional cells defined by distinctive surface markers and transcriptomes. This dataset enables flow cytometry solutions for in silico-predicted cell states and identifies dozens of cell surface markers consistently detected across donors spanning race and sex. Finally, aligning annotations from this atlas, we nominate normal marrow equivalents for acute myeloid leukemia stem cell populations that differ in clinical response. This atlas serves as an advanced digital resource for hematopoietic progenitor analyses in human health and disease.
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Células Madre Hematopoyéticas , Transcriptoma , Humanos , Médula Ósea , Perfilación de la Expresión Génica , Células de la Médula ÓseaRESUMEN
The role of splicing dysregulation in cancer is underscored by splicing factor mutations; however, its impact in the absence of such rare mutations is poorly understood. To reveal complex patient subtypes and putative regulators of pathogenic splicing in Acute Myeloid Leukemia (AML), we developed a new approach called OncoSplice. Among diverse new subtypes, OncoSplice identified a biphasic poor prognosis signature that partially phenocopies U2AF1-mutant splicing, impacting thousands of genes in over 40% of adult and pediatric AML cases. U2AF1-like splicing co-opted a healthy circadian splicing program, was stable over time and induced a leukemia stem cell (LSC) program. Pharmacological inhibition of the implicated U2AF1-like splicing regulator, PRMT5, rescued leukemia mis-splicing and inhibited leukemic cell growth. Genetic deletion of IRAK4, a common target of U2AF1-like and PRMT5 treated cells, blocked leukemia development in xenograft models and induced differentiation. These analyses reveal a new prognostic alternative-splicing mechanism in malignancy, independent of splicing-factor mutations.
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Immunotherapy has emerged as a crucial strategy to combat cancer by "reprogramming" a patient's own immune system. Although immunotherapy is typically reserved for patients with a high mutational burden, neoantigens produced from posttranscriptional regulation may provide an untapped reservoir of common immunogenic targets for new targeted therapies. To comprehensively define tumor-specific and likely immunogenic neoantigens from patient RNA-Seq, we developed Splicing Neo Antigen Finder (SNAF), an easy-to-use and open-source computational workflow to predict splicing-derived immunogenic MHC-bound peptides (T cell antigen) and unannotated transmembrane proteins with altered extracellular epitopes (B cell antigen). This workflow uses a highly accurate deep learning strategy for immunogenicity prediction (DeepImmuno) in conjunction with new algorithms to rank the tumor specificity of neoantigens (BayesTS) and to predict regulators of mis-splicing (RNA-SPRINT). T cell antigens from SNAF were frequently evidenced as HLA-presented peptides from mass spectrometry (MS) and predict response to immunotherapy in melanoma. Splicing neoantigen burden was attributed to coordinated splicing factor dysregulation. Shared splicing neoantigens were found in up to 90% of patients with melanoma, correlated to overall survival in multiple cancer cohorts, induced T cell reactivity, and were characterized by distinct cells of origin and amino acid preferences. In addition to T cell neoantigens, our B cell focused pipeline (SNAF-B) identified a new class of tumor-specific extracellular neoepitopes, which we termed ExNeoEpitopes. ExNeoEpitope full-length mRNA predictions were tumor specific and were validated using long-read isoform sequencing and in vitro transmembrane localization assays. Therefore, our systematic identification of splicing neoantigens revealed potential shared targets for therapy in heterogeneous cancers.
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Melanoma , Neoplasias , Humanos , Antígenos de Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/terapia , Linfocitos T , Péptidos/química , Inmunoterapia/métodosRESUMEN
Clinical outcome of patients with acute myeloid leukemia (AML) is associated with demographic and genetic features. Although the associations of acquired genetic alterations with patients' sex have been recently analyzed, their impact on outcome of female and male patients has not yet been comprehensively assessed. We performed mutational profiling, cytogenetic and outcome analyses in 1726 adults with AML (749 female and 977 male) treated on frontline Alliance for Clinical Trials in Oncology protocols. A validation cohort comprised 465 women and 489 men treated on frontline protocols of the German AML Cooperative Group. Compared with men, women more often had normal karyotype, FLT3-ITD, DNMT3A, NPM1 and WT1 mutations and less often complex karyotype, ASXL1, SRSF2, U2AF1, RUNX1, or KIT mutations. More women were in the 2022 European LeukemiaNet intermediate-risk group and more men in adverse-risk group. We found sex differences in co-occurring mutation patterns and prognostic impact of select genetic alterations. The mutation-associated splicing events and gene-expression profiles also differed between sexes. In patients aged <60 years, SF3B1 mutations were male-specific adverse outcome prognosticators. We conclude that sex differences in AML-associated genetic alterations and mutation-specific differential splicing events highlight the importance of patients' sex in analyses of AML biology and prognostication.
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Leucemia Mieloide Aguda , Caracteres Sexuales , Adulto , Humanos , Masculino , Femenino , Pronóstico , Nucleofosmina , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/tratamiento farmacológico , Mutación , Tirosina Quinasa 3 Similar a fms/genéticaRESUMEN
The dynamics of the hematopoietic flux responsible for blood cell production in native conditions remains a matter of debate. Using CITE-seq analyses, we uncovered a distinct progenitor population that displays a cell cycle gene signature similar to the one found in quiescent hematopoietic stem cells. We further determined that the CD62L marker can be used to phenotypically enrich this population in the Flt3+ multipotent progenitor (MPP4) compartment. Functional in vitro and in vivo analyses validated the heterogeneity of the MPP4 compartment and established the quiescent/slow-cycling properties of the CD62L- MPP4 cells. Furthermore, studies under native conditions revealed a novel hierarchical organization of the MPP compartments in which quiescent/slow-cycling MPP4 cells sustain a prolonged hematopoietic activity at steady-state while giving rise to other lineage-biased MPP populations. Altogether, our data characterize a durable and productive quiescent/slow-cycling hematopoietic intermediary within the MPP4 compartment and highlight early paths of progenitor differentiation during unperturbed hematopoiesis.
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Hematopoyesis , Células Madre Hematopoyéticas , Diferenciación Celular , División Celular , Células Madre MultipotentesRESUMEN
Radiation therapy (RT) is a crucial treatment modality for central nervous system (CNS) tumors but toxicity to healthy CNS tissues remains a challenge. Additionally, environmental exposure to radiation during nuclear catastrophes or space travel presents a risk of CNS toxicity. However, the underlying mechanisms of radiation-induced CNS toxicity are not fully understood. Neural progenitor cells (NPCs) are highly radiosensitive, resulting in decreased neurogenesis in the hippocampus. This study aimed to characterize a novel platform utilizing rat NPCs cultured as 3D neurospheres (NSps) to screen the safety and efficacy of experimental drugs with and without radiation exposure. The effect of radiation on NSp growth and differentiation was assessed by measuring sphere volume and the expression of neuronal differentiation markers Nestin and GFAP and proliferation marker Ki67. Radiation exposure inhibited NSp growth, decreased proliferation, and increased GFAP expression, indicating astrocytic differentiation. RNA sequencing analysis supported these findings, showing upregulation of Notch, BMP2/4, S100b, and GFAP gene expression during astrogenesis. By recapitulating radiation-induced toxicity and astrocytic differentiation, this single-NSp culture system provides a high-throughput preclinical model for assessing the effects of various radiation modalities and evaluates the safety and efficacy of potential therapeutic interventions in combination with radiation.
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Affinity-matured plasma cells (PCs) of varying lifespans are generated through a germinal center (GC) response. The developmental dynamics and genomic programs of antigen-specific PC precursors remain to be elucidated. Using a model antigen, we demonstrate biphasic generation of PC precursors, with those generating long-lived bone marrow PCs preferentially produced in the late phase of GC response. Clonal tracing using scRNA-seq+BCR-seq in spleen and bone marrow compartments, coupled with adoptive transfer experiments, reveal a novel PC transition state that gives rise to functionally competent PC precursors. The latter undergo clonal expansion, dependent on inducible expression of TIGIT. We propose a model for the proliferation and programming of precursors of long-lived PCs, based on extended antigen encounters followed by reduced antigen availability.
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There are many well-established relationships between pathogens and human disease, but far fewer when focusing on non-communicable diseases (NCDs). We leverage data from The UK Biobank and TriNetX to perform a systematic survey across 20 pathogens and 426 diseases, focused primarily on NCDs. To this end, we assess the association between disease status and infection history proxies. We identify 206 pathogen-disease pairs that replicate in both cohorts. We replicate many established relationships, including Helicobacter pylori with several gastroenterological diseases, and connections between Epstein-Barr virus with multiple sclerosis and lupus. Overall, our approach identified evidence of association for 15 of the pathogens and 96 distinct diseases, including a currently controversial link between human cytomegalovirus (CMV) and ulcerative colitis (UC). We validate this connection through two orthogonal analyses, revealing increased CMV gene expression in UC patients and enrichment for UC genetic risk signal near human genes that have altered expression upon CMV infection. Collectively, these results form a foundation for future investigations into mechanistic roles played by pathogens in disease.
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How two-chambered hearts in basal vertebrates have evolved from single-chamber hearts found in ancestral chordates remains unclear. Here, we show that the teleost sinus venosus (SV) is a chamber-like vessel comprised of an outer layer of smooth muscle cells. We find that in adult zebrafish nr2f1a mutants, which lack atria, the SV comes to physically resemble the thicker bulbus arteriosus (BA) at the arterial pole of the heart through an adaptive, hypertensive response involving smooth muscle proliferation due to aberrant hemodynamic flow. Single cell transcriptomics show that smooth muscle and endothelial cell populations within the adapting SV also take on arterial signatures. Bulk transcriptomics of the blood sinuses flanking the tunicate heart reinforce a model of greater equivalency in ancestral chordate BA and SV precursors. Our data simultaneously reveal that secondary complications from congenital heart defects can develop in adult zebrafish similar to those in humans and that the foundation of equivalency between flanking auxiliary vessels may remain latent within basal vertebrate hearts.
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Enfermedades Cardiovasculares , Cordados , Adulto , Humanos , Animales , Pez Cebra/genética , Aclimatación , Arterias , Atrios CardíacosRESUMEN
Systemic autoimmune and autoinflammatory diseases are characterized by genetic and cellular heterogeneity. While current single-cell genomics methods provide insights into known disease subtypes, these analysis methods do not readily reveal novel cell-type perturbation programs shared among distinct patient subsets. Here, we performed single-cell RNA-Seq of PBMCs of patients with systemic juvenile idiopathic arthritis (SJIA) with diverse clinical manifestations, including macrophage activation syndrome (MAS) and lung disease (LD). We introduced two new computational frameworks called UDON and SATAY-UDON, which define patient subtypes based on their underlying disrupted cellular programs as well as associated biomarkers or clinical features. Among twelve independently identified subtypes, this analysis uncovered what we believe to be a novel complement and interferon activation program identified in SJIA-LD monocytes. Extending these analyses to adult and pediatric lupus patients found new but also shared disease programs with SJIA, including interferon and complement activation. Finally, supervised comparison of these programs in a compiled single-cell pan-immune atlas of over 1,000 healthy donors found a handful of normal healthy donors with evidence of early inflammatory activation in subsets of monocytes and platelets, nominating possible biomarkers for early disease detection. Thus, integrative pan-immune single-cell analysis resolved what we believe to be new conserved gene programs underlying inflammatory disease pathogenesis and associated complications.
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Artritis Juvenil , Enfermedades Pulmonares , Adulto , Humanos , Niño , Artritis Juvenil/genética , Artritis Juvenil/complicaciones , Biomarcadores , Interferones , GenómicaRESUMEN
The transcription factor ThPOK (encoded by Zbtb7b) is well known for its role as a master regulator of CD4 lineage commitment in the thymus. Here, we report an unexpected and critical role of ThPOK as a multifaceted regulator of myeloid lineage commitment, differentiation and maturation. Using reporter and knockout mouse models combined with single-cell RNA-sequencing, progenitor transfer and colony assays, we show that ThPOK controls monocyte-dendritic cell versus granulocyte lineage production during homeostatic differentiation, and serves as a brake for neutrophil maturation in granulocyte lineage-specified cells through transcriptional regulation of lineage-specific transcription factors and RNA via altered messenger RNA splicing to reprogram intron retention.
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Regulación de la Expresión Génica , Timo , Animales , Ratones , Diferenciación Celular , Linaje de la Célula , Proteínas de Unión al ADN , Ratones Noqueados , ARN , Factores de Transcripción/genética , Antígenos CD4RESUMEN
Accurate cell type identification is a key and rate-limiting step in single-cell data analysis. Single-cell references with comprehensive cell types, reproducible and functionally validated cell identities, and common nomenclatures are much needed by the research community for automated cell type annotation, data integration, and data sharing. Here, we develop a computational pipeline utilizing the LungMAP CellCards as a dictionary to consolidate single-cell transcriptomic datasets of 104 human lungs and 17 mouse lung samples to construct LungMAP single-cell reference (CellRef) for both normal human and mouse lungs. CellRefs define 48 human and 40 mouse lung cell types catalogued from diverse anatomic locations and developmental time points. We demonstrate the accuracy and stability of LungMAP CellRefs and their utility for automated cell type annotation of both normal and diseased lungs using multiple independent methods and testing data. We develop user-friendly web interfaces for easy access and maximal utilization of the LungMAP CellRefs.
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Perfilación de la Expresión Génica , Difusión de la Información , Animales , Ratones , Humanos , Análisis de la Célula Individual , TranscriptomaRESUMEN
Acute myeloid leukemia (AML) is an aggressive blood cancer that stems from the rapid expansion of immature leukemic blasts in the bone marrow. Mutations in epigenetic factors represent the largest category of genetic drivers of AML. The chromatin assembly factor CHAF1B is a master epigenetic regulator of transcription associated with self-renewal and the undifferentiated state of AML blasts. Upregulation of CHAF1B, as observed in almost all AML samples, promotes leukemic progression by repressing the transcription of differentiation factors and tumor suppressors. However, the specific factors regulated by CHAF1B and their contributions to leukemogenesis are unstudied. We analyzed RNA sequencing data from mouse MLL-AF9 leukemic cells and bone marrow aspirates, representing a diverse collection of pediatric AML samples and identified the E3 ubiquitin ligase TRIM13 as a target of CHAF1B-mediated transcriptional repression associated with leukemogenesis. We found that CHAF1B binds the promoter of TRIM13, resulting in its transcriptional repression. In turn, TRIM13 suppresses self-renewal of leukemic cells by promoting pernicious entry into the cell cycle through its nuclear localization and catalytic ubiquitination of cell cycle-promoting protein, CCNA1. Overexpression of TRIM13 initially prompted a proliferative burst in AML cells, which was followed by exhaustion, whereas loss of total TRIM13 or deletion of its catalytic domain enhanced leukemogenesis in AML cell lines and patient-derived xenografts. These data suggest that CHAF1B promotes leukemic development, in part, by repressing TRIM13 expression and that this relationship is necessary for leukemic progression.
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Ensamble y Desensamble de Cromatina , Leucemia Mieloide Aguda , Humanos , Ratones , Animales , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Línea Celular , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Factor 1 de Ensamblaje de la Cromatina/genética , Factor 1 de Ensamblaje de la Cromatina/metabolismo , Proteínas de Unión al ADN/genética , Proteínas Supresoras de Tumor/metabolismo , Proteínas de Motivos Tripartitos/genética , Proteínas de Motivos Tripartitos/metabolismoRESUMEN
Clinical evidence points to a function for B cell-activating factor (BAFF) in pregnancy. However, direct roles for BAFF-axis members in pregnancy have not been examined. Here, via utility of genetically modified mice, we report that BAFF promotes inflammatory responsiveness and increases susceptibility to inflammation-induced preterm birth (PTB). In contrast, we show that the closely related A proliferation-inducing ligand (APRIL) decreases inflammatory responsiveness and susceptibility to PTB. Known BAFF-axis receptors serve a redundant function in signaling BAFF/APRIL presence in pregnancy. Treatment with anti-BAFF/APRIL monoclonal antibodies or BAFF/APRIL recombinant proteins is sufficient to manipulate susceptibility to PTB. Notably, macrophages at the maternal-fetal interface produce BAFF, while BAFF and APRIL presence divergently shape macrophage gene expression and inflammatory function. Overall, our findings demonstrate that BAFF and APRIL play divergent inflammatory roles in pregnancy and provide therapeutic targets for mitigating risk of inflammation-induced PTB.
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Nacimiento Prematuro , Animales , Femenino , Ratones , Embarazo , Factor Activador de Células B , Inflamación , Transducción de Señal , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/genéticaRESUMEN
MOTIVATION: While conventional flow cytometry is limited to dozens of markers, new experimental and computational strategies, such as Infinity Flow, allow for the generation and imputation of hundreds of cell surface protein markers in millions of cells. Here, we describe an end-to-end analysis workflow for Infinity Flow data in Python. RESULTS: pyInfinityFlow enables the efficient analysis of millions of cells, without down-sampling, through direct integration with well-established Python packages for single-cell genomics analysis. pyInfinityFlow accurately identifies both common and extremely rare cell populations which are challenging to define from single-cell genomics studies alone. We demonstrate that this workflow can nominate novel markers to design new flow cytometry gating strategies for predicted cell populations. pyInfinityFlow can be extended to diverse cell discovery analyses with flexibility to adapt to diverse Infinity Flow experimental designs. AVAILABILITY AND IMPLEMENTATION: pyInfinityFlow is freely available in GitHub (https://github.com/KyleFerchen/pyInfinityFlow) and on PyPI (https://pypi.org/project/pyInfinityFlow/). Package documentation with tutorials on a test dataset is available by Read the Docs (pyinfinityflow.readthedocs.io). The scripts and data for reproducing the results are available at https://github.com/KyleFerchen/pyInfinityFlow/tree/main/analysis_scripts, along with the raw flow cytometry input data.