Molecular assessment of intratumoral immune cell subsets and potential mechanisms of resistance to odronextamab, a CD20×CD3 bispecific antibody, in patients with relapsed/refractory B-cell non-Hodgkin lymphoma.

BACKGROUND: Patients with relapsed/refractory B-cell non-Hodgkin lymphoma (R/R B-NHL) have a significant need for effective treatment options. Odronextamab is an Fc-silenced, human, CD20×CD3 bispecific antibody that targets CD20-expressing cells via T-cell-mediated cytotoxicity independent of T-cell/major histocompatibility complex interaction. Phase I results in patients with R/R B-NHL demonstrated that odronextamab monotherapy could achieve deep and durable responses with a generally manageable safety profile (ELM-1; NCT02290951). As part of a biomarker analysis of the same study, we investigated potential biomarkers and mechanisms of resistance to odronextamab. METHODS: Patients with R/R B-NHL enrolled in ELM-1 received one time per week doses of intravenous odronextamab for 4×21 day cycles, then doses every 2 weeks thereafter. Patient tumor biopsies were obtained at baseline, on-treatment, and at progression. Immune cell markers were analyzed by immunohistochemistry, flow cytometry, single-cell RNA sequencing, and whole genome sequencing. RESULTS: Baseline tumor biopsies showed that almost all patients had high proportions of B cells that expressed the CD20 target antigen, whereas expression of other B-cell surface antigens (CD19, CD22, CD79b) was more variable. Responses to odronextamab in patients with diffuse large B-cell lymphoma were not related to the relative level of baseline CD20 expression, cell of origin, or high-risk molecular subtype. A potential link was observed between greater tumor programmed cell death-ligand 1 expression and increased likelihood of response to odronextamab. Similarly, a trend was observed between clinical response and increased levels of CD8 T cells and regulatory T cells at baseline. We also identified an on-treatment pharmacodynamic shift in intratumoral immune cell subsets. Finally, loss of CD20 expression through inactivating gene mutations was identified as a potential mechanism of resistance in patients who were treated with odronextamab until progression, as highlighted in two detailed patient cases reported here. CONCLUSIONS: This biomarker analysis expands on clinical findings of odronextamab in patients with R/R B-NHL, providing verification of the suitability of CD20 as a therapeutic target, as well as evidence for potential mechanisms of action and resistance.

Perturbed liver gene zonation in a mouse model of non-alcoholic steatohepatitis.

Liver zonation characterizes the separation of metabolic pathways along the lobules and is required for optimal hepatic function. Wnt signaling is a master regulator of spatial liver zonation. A perivenous-periportal Wnt activity gradient orchestrates metabolic zonation by activating gene expression in perivenous hepatocytes, while suppressing gene expression in their periportal counterparts. However, the understanding as to the liver gene zonation and zonation regulators in diseases is limited. Non-alcoholic steatohepatitis (NASH) is a chronic liver disease characterized by fat accumulation, inflammation, and fibrosis. Here, we investigated the perturbation of liver gene zonation in a mouse NASH model by combining spatial transcriptomics, bulk RNAseq and in situ hybridization. Wnt-target genes represented a major subset of genes showing altered spatial expression in the NASH liver. The altered Wnt-target gene expression levels and zonation spatial patterns were in line with the up regulation of Wnt regulators and the augmentation of Wnt signaling. Particularly, we found that the Wnt activator Rspo3 expression was restricted to the perivenous zone in control liver but expanded to the periportal zone in NASH liver. AAV8-mediated RSPO3 overexpression in controls resulted in zonation changes, and further amplified the disturbed zonation of Wnt-target genes in NASH, similarly Rspo3 knockdown in Rspo3+/- mice resulted in zonation changes of Wnt-target genes in both chow and HFD mouse. Interestingly, there were no impacts on steatosis, inflammation, or fibrosis NASH pathology from RSPO3 overexpression nor Rspo3 knockdown. In summary, our study demonstrated the alteration of Wnt signaling in a mouse NASH model, leading to perturbed liver zonation.

Ancient dental calculus reveals oral microbiome shifts associated with lifestyle and disease in Great Britain.

The prevalence of chronic, non-communicable diseases has risen sharply in recent decades, especially in industrialized countries. While several studies implicate the microbiome in this trend, few have examined the evolutionary history of industrialized microbiomes. Here we sampled 235 ancient dental calculus samples from individuals living in Great Britain (∼2200 BCE to 1853 CE), including 127 well-contextualized London adults. We reconstructed their microbial history spanning the transition to industrialization. After controlling for oral geography and technical biases, we identified multiple oral microbial communities that coexisted in Britain for millennia, including a community associated with Methanobrevibacter, an anaerobic Archaea not commonly prevalent in the oral microbiome of modern industrialized societies. Calculus analysis suggests that oral hygiene contributed to oral microbiome composition, while microbial functions reflected past differences in diet, specifically in dairy and carbohydrate consumption. In London samples, Methanobrevibacter-associated microbial communities are linked with skeletal markers of systemic diseases (for example, periostitis and joint pathologies), and their disappearance is consistent with temporal shifts, including the arrival of the Second Plague Pandemic. This suggests pre-industrialized microbiomes were more diverse than previously recognized, enhancing our understanding of chronic, non-communicable disease origins in industrialized populations.

TRAPnSeq allows high-throughput profiling of antigen-specific antibody-secreting cells.

Following activation by cognate antigen, B cells undergo fine-tuning of their antigen receptors and may ultimately differentiate into antibody-secreting cells (ASCs). While antigen-specific B cells that express surface receptors (B cell receptors [BCRs]) can be readily cloned and sequenced following flow sorting, antigen-specific ASCs that lack surface BCRs cannot be easily profiled. Here, we report an approach, TRAPnSeq (antigen specificity mapping through immunoglobulin [Ig] secretion TRAP and Sequencing), that allows capture of secreted antibodies on the surface of ASCs, which in turn enables high-throughput screening of single ASCs against large antigen panels. This approach incorporates flow cytometry, standard microfluidic platforms, and DNA-barcoding technologies to characterize antigen-specific ASCs through single-cell V(D)J, RNA, and antigen barcode sequencing. We show the utility of TRAPnSeq by profiling antigen-specific IgG and IgE ASCs from both mice and humans and highlight its capacity to accelerate therapeutic antibody discovery from ASCs.

A cellular and molecular spatial atlas of dystrophic muscle.

Asynchronous skeletal muscle degeneration/regeneration is a hallmark feature of Duchenne muscular dystrophy (DMD); however, traditional -omics technologies that lack spatial context make it difficult to study the biological mechanisms of how asynchronous regeneration contributes to disease progression. Here, using the severely dystrophic D2-mdx mouse model, we generated a high-resolution cellular and molecular spatial atlas of dystrophic muscle by integrating spatial transcriptomics and single-cell RNAseq datasets. Unbiased clustering revealed nonuniform distribution of unique cell populations throughout D2-mdx muscle that were associated with multiple regenerative timepoints, demonstrating that this model faithfully recapitulates the asynchronous regeneration observed in human DMD muscle. By probing spatiotemporal gene expression signatures, we found that propagation of inflammatory and fibrotic signals from locally damaged areas contributes to widespread pathology and that querying expression signatures within discrete microenvironments can identify targetable pathways for DMD therapy. Overall, this spatial atlas of dystrophic muscle provides a valuable resource for studying DMD disease biology and therapeutic target discovery.

Intratumoral dendritic cell-CD4+ T helper cell niches enable CD8+ T cell differentiation following PD-1 blockade in hepatocellular carcinoma.

Despite no apparent defects in T cell priming and recruitment to tumors, a large subset of T cell rich tumors fail to respond to immune checkpoint blockade (ICB). We leveraged a neoadjuvant anti-PD-1 trial in patients with hepatocellular carcinoma (HCC), as well as additional samples collected from patients treated off-label, to explore correlates of response to ICB within T cell-rich tumors. We show that ICB response correlated with the clonal expansion of intratumoral CXCL13+CH25H+IL-21+PD-1+CD4+ T helper cells ("CXCL13+ TH") and Granzyme K+ PD-1+ effector-like CD8+ T cells, whereas terminally exhausted CD39hiTOXhiPD-1hiCD8+ T cells dominated in nonresponders. CD4+ and CD8+ T cell clones that expanded post-treatment were found in pretreatment biopsies. Notably, PD-1+TCF-1+ (Progenitor-exhausted) CD8+ T cells shared clones mainly with effector-like cells in responders or terminally exhausted cells in nonresponders, suggesting that local CD8+ T cell differentiation occurs upon ICB. We found that these Progenitor CD8+ T cells interact with CXCL13+ TH within cellular triads around dendritic cells enriched in maturation and regulatory molecules, or "mregDC". These results suggest that discrete intratumoral niches that include mregDC and CXCL13+ TH control the differentiation of tumor-specific Progenitor exhasuted CD8+ T cells following ICB.

Rapid TCR:Epitope Ranker (RAPTER): a primary human T cell reactivity screening assay pairing epitope and TCR at single cell resolution.

Identifying epitopes that T cells respond to is critical for understanding T cell-mediated immunity. Traditional multimer and other single cell assays often require large blood volumes and/or expensive HLA-specific reagents and provide limited phenotypic and functional information. Here, we present the Rapid TCR:Epitope Ranker (RAPTER) assay, a single cell RNA sequencing (scRNA-SEQ) method that uses primary human T cells and antigen presenting cells (APCs) to assess functional T cell reactivity. Using hash-tag oligonucleotide (HTO) coding and T cell activation-induced markers (AIM), RAPTER defines paired epitope specificity and TCR sequence and can include RNA- and protein-level T cell phenotype information. We demonstrate that RAPTER identified specific reactivities to viral and tumor antigens at sensitivities as low as 0.15% of total CD8+ T cells, and deconvoluted low-frequency circulating HPV16-specific T cell clones from a cervical cancer patient. The specificities of TCRs identified by RAPTER for MART1, EBV, and influenza epitopes were functionally confirmed in vitro. In summary, RAPTER identifies low-frequency T cell reactivities using primary cells from low blood volumes, and the resulting paired TCR:ligand information can directly enable immunogenic antigen selection from limited patient samples for vaccine epitope inclusion, antigen-specific TCR tracking, and TCR cloning for further therapeutic development.

Spontaneous tumor regression mediated by human T cells in a humanized immune system mouse model.

Immunodeficient mice reconstituted with a human immune system (HIS mice) give rise to human T cells, which make them an attractive system to study human immune responses to tumors. However, such HIS mice typically exhibit sub-optimal responses to immune challenges as well as fail to develop antigen-specific B or T cell memory. Here we report HIS mice mediate spontaneous regression of human B cell lymphoma Raji. Tumor regression was dependent on CD4+ and CD8+ T cell responses and resulted in T cell memory. The T cell memory elicited was mainly Raji-specific, however some level of cross-protection was also elicited to a related B cell lymphoma cell line Ramos. Single-cell RNAseq analysis indicated activation of CD8+ T cells in regressing Raji tumors as well as clonal expansion of specific T cell receptors (TCRs). Cloning of TCRs from Raji-infiltrating T cells into a Jurkat reporter cell line showed reactivity specific for Raji tumor cells. Overall, we report a platform for studying in vivo human T cell tumor immunity by highlighting spontaneous Raji tumor regression, clonal TCR expansion, and T cell memory in HIS mice.

Unlocking Modifiable Risk Factors for Alzheimer's Disease: Does the Oral Microbiome Hold Some of the Keys?

Advancing age is recognized as the primary risk factor for Alzheimer's disease (AD); however approximately one third of dementia cases are attributable to modifiable risk factors such as hypertension, diabetes, smoking, and obesity. Recent research also implicates oral health and the oral microbiome in AD risk and pathophysiology. The oral microbiome contributes to the cerebrovascular and neurodegenerative pathology of AD via the inflammatory, vascular, neurotoxic, and oxidative stress pathways of known modifiable risk factors. This review proposes a conceptual framework that integrates the emerging evidence regarding the oral microbiome with established modifiable risk factors. There are numerous mechanisms by which the oral microbiome may interact with AD pathophysiology. Microbiota have immunomodulatory functions, including the activation of systemic pro-inflammatory cytokines. This inflammation can affect the integrity of the blood-brain barrier, which in turn modulates translocation of bacteria and their metabolites to brain parenchyma. Amyloid-ß is an antimicrobial peptide, a feature which may in part explain its accumulation. There are microbial interactions with cardiovascular health, glucose tolerance, physical activity, and sleep, suggesting that these modifiable lifestyle risk factors of dementia may have microbial contributors. There is mounting evidence to suggest the relevance of oral health practices and the microbiome to AD. The conceptual framework presented here additionally demonstrates the potential for the oral microbiome to comprise a mechanistic intermediary between some lifestyle risk factors and AD pathophysiology. Future clinical studies may identify specific oral microbial targets and the optimum oral health practices to reduce dementia risk.

Development of the oral resistome during the first decade of life.

Antibiotic overuse has promoted the spread of antimicrobial resistance (AMR) with significant health and economic consequences. Genome sequencing reveals the widespread presence of antimicrobial resistance genes (ARGs) in diverse microbial environments. Hence, surveillance of resistance reservoirs, like the rarely explored oral microbiome, is necessary to combat AMR. Here, we characterise the development of the paediatric oral resistome and investigate its role in dental caries in 221 twin children (124 females and 97 males) sampled at three time points over the first decade of life. From 530 oral metagenomes, we identify 309 ARGs, which significantly cluster by age, with host genetic effects detected from infancy onwards. Our results suggest potential mobilisation of ARGs increases with age as the AMR associated mobile genetic element, Tn916 transposase was co-located with more species and ARGs in older children. We find a depletion of ARGs and species in dental caries compared to health. This trend reverses in restored teeth. Here we show the paediatric oral resistome is an inherent and dynamic component of the oral microbiome, with a potential role in transmission of AMR and dysbiosis.

Cancer cell-derived type I interferons instruct tumor monocyte polarization.

Monocytes are highly plastic immune cells that modulate antitumor immunity. Therefore, identifying factors that regulate tumor monocyte functions is critical for developing effective immunotherapies. Here, we determine that endogenous cancer cell-derived type I interferons (IFNs) control monocyte functional polarization. Guided by single-cell transcriptomic profiling of human and mouse tumors, we devise a strategy to distinguish and separate immunostimulatory from immunosuppressive tumor monocytes by surface CD88 and Sca-1 expression. Leveraging this approach, we show that cGAS-STING-regulated cancer cell-derived IFNs polarize immunostimulatory monocytes associated with anti-PD-1 immunotherapy response in mice. We also demonstrate that immunosuppressive monocytes convert into immunostimulatory monocytes upon cancer cell-intrinsic cGAS-STING activation. Consistently, we find that human cancer cells can produce type I IFNs that polarize monocytes, and our immunostimulatory monocyte gene signature is enriched in patient tumors that respond to anti-PD-1 immunotherapy. Our work exposes a role for cancer cell-derived IFNs in licensing monocyte functions that influence immunotherapy outcomes.

A single-cell map of dynamic chromatin landscapes of immune cells in renal cell carcinoma.

A complete chart of the chromatin regulatory elements of immune cells in patients with cancer and their dynamic behavior is necessary to understand the developmental fates and guide therapeutic strategies. Here, we map the single-cell chromatin landscape of immune cells from blood, normal tumor-adjacent kidney tissue and malignant tissue from patients with early-stage clear cell renal cell carcinoma (ccRCC). We catalog the T cell states dictated by tissue-specific and developmental-stage-specific chromatin accessibility patterns, infer key chromatin regulators and observe rewiring of regulatory networks in the progression to dysfunction in CD8+ T cells. Unexpectedly, among the transcription factors orchestrating the path to dysfunction, NF-κB is associated with a pro-apoptotic program in late stages of dysfunction in tumor-infiltrating CD8+ T cells. Importantly, this epigenomic profiling stratified ccRCC patients based on a NF-κB-driven pro-apoptotic signature. This study provides a rich resource for understanding the functional states and regulatory dynamics of immune cells in ccRCC.

A randomized clinical trial to investigate the effect of dietary protein sources on periodontal health.

AIM: The aim was to assess two macronutrient interventions in a 2 × 2 factorial dietary design to determine their effects on oral health. MATERIALS AND METHODS: Participants (65-75 years old) with a body mass index between 20 and 35 kg/m2 of a larger randomized control trial who consented to an oral health assessment were recruited. They had ad libitum access to one of four experimental diets (omnivorous higher fat or higher carbohydrate, semi-vegetarian higher fat or higher carbohydrate) for 4 weeks. The periodontal examination included periodontal probing depth (PPD), clinical attachment level (CAL), and bleeding on probing. Oral plaque and gingival crevicular fluid (GCF) were collected before and after the intervention. RESULTS: Between baseline and follow up, the number of sites with a CAL <5 mm (mean difference [MD] -5.11 ± 9.68, p = .039) increased and the GCF amount (MD -23.42 ± 39.42 Periotron Units [PU], p = .050) decreased for the semi-vegetarian high-fat diet. For the mean proportion of sites with PPD reduction of >1 mm and CAL gain of >1 mm, significant differences were calculated between the diets investigated. The clinical parameters were not associated with changes in the oral microbiota. CONCLUSIONS: The results of this study provided evidence that a semi-vegetarian high-fat diet provides benefits to clinical parameters of periodontal health. This study is registered in ClinicalTrials.gov (ACTRN12616001606471).

AdRoit is an accurate and robust method to infer complex transcriptome composition.

Bulk RNA sequencing provides the opportunity to understand biology at the whole transcriptome level without the prohibitive cost of single cell profiling. Advances in spatial transcriptomics enable to dissect tissue organization and function by genome-wide gene expressions. However, the readout of both technologies is the overall gene expression across potentially many cell types without directly providing the information of cell type constitution. Although several in-silico approaches have been proposed to deconvolute RNA-Seq data composed of multiple cell types, many suffer a deterioration of performance in complex tissues. Here we present AdRoit, an accurate and robust method to infer the cell composition from transcriptome data of mixed cell types. AdRoit uses gene expression profiles obtained from single cell RNA sequencing as a reference. It employs an adaptive learning approach to alleviate the sequencing technique difference between the single cell and the bulk (or spatial) transcriptome data, enhancing cross-platform readout comparability. Our systematic benchmarking and applications, which include deconvoluting complex mixtures that encompass 30 cell types, demonstrate its preferable sensitivity and specificity compared to many existing methods as well as its utilities. In addition, AdRoit is computationally efficient and runs orders of magnitude faster than most methods.

Molecular characteristics and spatial distribution of adult human corneal cell subtypes.

Bulk RNA sequencing of a tissue captures the gene expression profile from all cell types combined. Single-cell RNA sequencing identifies discrete cell-signatures based on transcriptomic identities. Six adult human corneas were processed for single-cell RNAseq and 16 cell clusters were bioinformatically identified. Based on their transcriptomic signatures and RNAscope results using representative cluster marker genes on human cornea cross-sections, these clusters were confirmed to be stromal keratocytes, endothelium, several subtypes of corneal epithelium, conjunctival epithelium, and supportive cells in the limbal stem cell niche. The complexity of the epithelial cell layer was captured by eight distinct corneal clusters and three conjunctival clusters. These were further characterized by enriched biological pathways and molecular characteristics which revealed novel groupings related to development, function, and location within the epithelial layer. Moreover, epithelial subtypes were found to reflect their initial generation in the limbal region, differentiation, and migration through to mature epithelial cells. The single-cell map of the human cornea deepens the knowledge of the cellular subsets of the cornea on a whole genome transcriptional level. This information can be applied to better understand normal corneal biology, serve as a reference to understand corneal disease pathology, and provide potential insights into therapeutic approaches.

Butyrophilin-like 2 regulates site-specific adaptations of intestinal γδ intraepithelial lymphocytes.

Tissue-resident γδ intraepithelial lymphocytes (IELs) orchestrate innate and adaptive immune responses to maintain intestinal epithelial barrier integrity. Epithelia-specific butyrophilin-like (Btnl) molecules induce perinatal development of distinct Vγ TCR+ IELs, however, the mechanisms that control γδ IEL maintenance within discrete intestinal segments are unclear. Here, we show that Btnl2 suppressed homeostatic proliferation of γδ IELs preferentially in the ileum. High throughput transcriptomic characterization of site-specific Btnl2-KO γδ IELs reveals that Btnl2 regulated the antimicrobial response module of ileal γδ IELs. Btnl2 deficiency shapes the TCR specificities and TCRγ/δ repertoire diversity of ileal γδ IELs. During DSS-induced colitis, Btnl2-KO mice exhibit increased inflammation and delayed mucosal repair in the colon. Collectively, these data suggest that Btnl2 fine-tunes γδ IEL frequencies and TCR specificities in response to site-specific homeostatic and inflammatory cues. Hence, Btnl-mediated targeting of γδ IEL development and maintenance may help dissect their immunological functions in intestinal diseases with segment-specific manifestations.

How does the early life environment influence the oral microbiome and determine oral health outcomes in childhood?

The first 1000 days of life, from conception to 2 years, are a critical window for the influence of environmental exposures on the assembly of the oral microbiome, which is the precursor to dental caries (decay), one of the most prevalent microbially induced disorders worldwide. While it is known that the human microbiome is susceptible to environmental exposures, there is limited understanding of the impact of prenatal and early childhood exposures on the oral microbiome trajectory and oral health. A barrier has been the lack of technology to directly measure the foetal "exposome", which includes nutritional and toxic exposures crossing the placenta. Another barrier has been the lack of statistical methods to account for the high dimensional data generated by-omic assays. Through identifying which early life exposures influence the oral microbiome and modify oral health, these findings can be translated into interventions to reduce dental decay prevalence.

Endogenous retroviral proteins provide an immunodominant but not requisite antigen in a murine immunotherapy tumor model.

Clinical observations suggest that responses to cancer immunotherapy are correlated with intra-tumoral T cell receptor (TCR) clonality, tumor mutation burden (TMB) and host HLA genotype, highlighting the importance of host T cell recognition of tumor antigens. However, the dynamic interplay between T cell activation state and changes in TCR repertoire in driving the identification of potential immunodominant antigen(s) remains largely unexplored. Here, we performed single-cell RNA-sequencing on CD8+ tumor-infiltrating T cells (TILs) using the murine colorectal tumor model MC38 to identify unique TCR sequences and validate their tumor reactivity. We found that the majority of clonally expanded TILs are tumor-reactive and their TCR repertoire is unique amongst individual MC38 tumor-bearing mice. Our query identified that multiple expanded TCR clones recognized the retroviral epitope p15E as an immunodominant antigen. In addition, we found that the endogenous retroviral genome encoding for p15E is highly expressed in MC38 tumors, but not in normal tissues, due to epigenetic derepression. Further, we demonstrated that the p15E-specific TILs exhibit an activated phenotype and an increase in frequency upon treatment with anti-41BB and anti-PD-1 combination immunotherapy. Importantly, we showed that although p15E-specific TILs are not required to mount a primary anti-tumor response, they contributed to the development of strong immune memory. Overall our results revealed that endogenous retroviral antigens expressed by tumor cells may represent an important and underappreciated category of tumor antigens that could be readily targeted in the clinic.

Oral microbiome composition, but not diversity, is associated with adolescent anxiety and depression symptoms.

PURPOSE: Depression and anxiety are highly prevalent disorders, whose significant burden is compounded by the presence of oral disease. Mental health disorders and oral health may be associated via changes to the oral microbiome, involving increased pro-inflammatory communication and cortisol in saliva. The present study provides the first culture-independent investigation of the oral microbiome considering depression and anxiety symptoms in adolescence, a critical age where these conditions begin to emerge and co-occur. It also investigates whether inflammation and cortisol moderate these relationships. METHODS: Participants (N = 66) aged 14-18 years (69.70% female) self-reported oral health, depression and anxiety symptoms, and collected saliva samples across two days. Saliva was assayed for cortisol and C-reactive protein (CRP), and used for 16S rRNA gene sequencing to estimate the oral microbiome. Multivariate statistical analyses examined associations. RESULTS: Overall diversity of the oral microbiome did not differ between adolescents by anxiety or depression grouping (low versus high symptoms), and was not associated with symptom measures. Depression and anxiety symptoms were instead associated with differential abundance of specific bacterial taxa, including Spirochaetaceae, Actinomyces, Treponema, Fusobacterium and Leptotrichia spp. Several host mood-microbial relationships were moderated by proposed mechanisms, including salivary cortisol and CRP. CONCLUSIONS: Oral microbiome composition, but not diversity, was associated with adolescent anxiety and depression symptoms. Longitudinal studies considering these associations would improve mechanistic understanding. This research indicates that adolescence remains an essential developmental period to identify early targets for intervention.

Molecular taxonomy of human ocular outflow tissues defined by single-cell transcriptomics.

The conventional outflow pathway is a complex tissue responsible for maintaining intraocular pressure (IOP) homeostasis. The coordinated effort of multiple cells with differing responsibilities ensures healthy outflow function and IOP maintenance. Dysfunction of one or more resident cell types results in ocular hypertension and risk for glaucoma, a leading cause of blindness. In this study, single-cell RNA sequencing was performed to generate a comprehensive cell atlas of human conventional outflow tissues. We obtained expression profiles of 17,757 genes from 8,758 cells from eight eyes of human donors representing the outflow cell transcriptome. Upon clustering analysis, 12 distinct cell types were identified, and region-specific expression of candidate genes was mapped in human tissues. Significantly, we identified two distinct expression patterns (myofibroblast- and fibroblast-like) from cells located in the trabecular meshwork (TM), the primary structural component of the conventional outflow pathway. We also located Schwann cell and macrophage signatures in the TM. The second primary component structure, Schlemm's canal, displayed a unique combination of lymphatic/blood vascular gene expression. Other expression clusters corresponded to cells from neighboring tissues, predominantly in the ciliary muscle/scleral spur, which together correspond to the uveoscleral outflow pathway. Importantly, the utility of our atlas was demonstrated by mapping glaucoma-relevant genes to outflow cell clusters. Our study provides a comprehensive molecular and cellular classification of conventional and unconventional outflow pathway structures responsible for IOP homeostasis.