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Jul 2018 • Cancer Research 78 (13 Supplement), 4679-4679, 2018

Rectal cancer-infiltrating T cells show clonal expansion associated with spatially restricted tolerogenic phenotypes

Livius Penter, Kerstin Dietze, Felix Aigner, Lars Bullinger, Thomas Blankenstein, Leo Hansmann

T cell infiltration correlates with prognosis and outcome in colorectal cancer regardless of the tumor stage. However, data on clonal expansion, phenotypes, functions, and spatial distribution of tumor-infiltrating T cells (TILs) are limited.We hypothesized that subsets of clonally expanded rectal cancer-associated T cells are specifically recruited into the tumor and show characteristic phenotypes and functions.Paired TILs and T cells from adjacent unaffected mucosa were isolated from five treatment-naïve rectal cancer patients. Although T cell immune phenotypes were heterogeneous, the frequencies of CD38+, PD-1+, and TIM-3+ cells were significantly higher in CD8+ TILs when compared to T cells from unaffected mucosa (p < 0.05). To track clonal expansion and phenotypes at the single cell level, we combined 13-parameter FACS single cell index sorting with next generation T cell receptor (TCR) and phenotype …

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Jul 2018 • Cancer Research 78 (13 Supplement), 1220-1220, 2018

Integrative genomic analysis discovers the causative regulatory mechanisms of a breast cancer-associated genetic variant

Yi Zhang, Mohith Manjunath, Shilu Zhang, Deborah Chasman, Sushmita Roy, Jun S Song

Genome-wide association studies (GWAS) have identified genetic variants that may significantly modulate breast cancer susceptibility. However, the precise molecular mechanisms behind these associations remain largely unknown; often, it is not even clear whether the GWAS variants are functional themselves or just genetically linked to other functional variants. We here provide an integrated method for identifying functional regulatory variants associated with breast cancer and their target genes by combining the analyses of expression quantitative trait loci (eQTL), a modified version of allele-specific expression (ASE) systematically utilizing haplotype information, transcription factor (TF) binding preference, and epigenetic information. Application of our method to the breast cancer susceptibility region in 5p12 demonstrates that the GWAS risk allele rs4415084-T is correlated with higher expression levels of the …

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Jul 2018 • European journal of immunology

FACS single cell index sorting is highly reliable and determines immune phenotypes of clonally expanded T cells

Livius Penter, Kerstin Dietze, Lars Bullinger, Jörg Westermann, Hans‐Peter Rahn, Leo Hansmann

FACS index sorting allows the isolation of single cells with retrospective identification of each single cell's high‐dimensional immune phenotype. We experimentally determine the error rate of index sorting and combine the technology with T cell receptor sequencing to identify clonal T cell expansion in aplastic anemia bone marrow as an example.

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Jul 2018 • Proceedings of the National Academy of Sciences

Estrogen-regulated feedback loop limits the efficacy of estrogen receptor–targeted breast cancer therapy

Tengfei Xiao, Wei Li, Xiaoqing Wang, Han Xu, Jixin Yang, Qiu Wu, Ying Huang, Joseph Geradts, Peng Jiang, Teng Fei, David Chi, Chongzhi Zang, Qi Liao, Jonathan Rennhack, Eran Andrechek, Nanlin Li, Simone Detre, Mitchell Dowsett, Rinath M Jeselsohn, X Shirley Liu, Myles Brown

Endocrine therapy resistance invariably develops in advanced estrogen receptor-positive (ER+) breast cancer, but the underlying mechanisms are largely unknown. We have identified C-terminal SRC kinase (CSK) as a critical node in a previously unappreciated negative feedback loop that limits the efficacy of current ER-targeted therapies. Estrogen directly drives CSK expression in ER+ breast cancer. At low CSK levels, as is the case in patients with ER+ breast cancer resistant to endocrine therapy and with the poorest outcomes, the p21 protein-activated kinase 2 (PAK2) becomes activated and drives estrogen-independent growth. PAK2 overexpression is also associated with endocrine therapy resistance and worse clinical outcome, and the combination of a PAK2 inhibitor with an ER antagonist synergistically suppressed breast tumor growth. Clinical approaches to endocrine therapy-resistant breast cancer …

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Jul 2018 • American Association for Cancer Research

Single-cell mass cytometry of classical Hodgkin lymphoma defines an exhausted and immunosuppressive microenvironment

Fathima Z Cader, Ron C Schackmann, Xihao Hu, Kirsty Wienand, Robert A Redd, Bjoern Chapuy, Jing Ouyang, Nicole E Paul, Evisa Gjini, Mikel Lipschitz, Laura M Selfors, Philippe Armand, David Wu, Jonathan R Fromm, Donna Neuberg, Xiaole S Liu, Scott J Rodig, Margaret A Shipp

Background: In classical Hodgkin lymphoma (cHL), the rare malignant Hodgkin Reed-Sternberg (HRS) cells are surrounded by an inflammatory infiltrate. Yet, the host anti-tumor immune response is ineffective. HRS cells have multifaceted mechanisms to evade the immune system including 9p24.1/PD-L1/PD-L2 genetic alterations leading to overexpression of PD-1 ligands and subsequent T cell exhaustion, aberrant antigen presentation and modulation of the tumor microenvironment (TME).The clinical success of PD-1 blockade in cHL suggests the TME contains reversibly exhausted T-effectors (Teff). Paradoxically, durable responses are observed in patients with β2M/MHC class I loss on HRS cells, raising the possibility of non-CD8+ mediated mechanisms of efficacy of PD-1 blockade. For this reason, we sought to characterize HRS cells and the surrounding TME.Methods: Using CyTOF technology, we evaluated …

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Jul 2018 • Annual Review of Biomedical Data Science 1, 1-27, 2018

Big Data Approaches for Modeling Response and Resistance to Cancer Drugs

Peng Jiang, William R Sellers, X Shirley Liu

Despite significant progress in cancer research, current standard-of-care drugs fail to cure many types of cancers. Hence, there is an urgent need to identify better predictive biomarkers and treatment regimes. Conventionally, insights from hypothesis-driven studies are the primary force for cancer biology and therapeutic discoveries. Recently, the rapid growth of big data resources, catalyzed by breakthroughs in high-throughput technologies, has resulted in a paradigm shift in cancer therapeutic research. The combination of computational methods and genomics data has led to several successful clinical applications. In this review, we focus on recent advances in data-driven methods to model anticancer drug efficacy, and we present the challenges and opportunities for data science in cancer therapeutic research.

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Jun 2018 • Bioinformatics

Improved design and analysis of CRISPR knockout screens

Chen-Hao Chen, Tengfei Xiao, Han Xu, Peng Jiang, Clifford A Meyer, Wei Li, Myles Brown, X Shirley Liu

MotivationGenome-wide clustered, regularly interspaced, short palindromic repeat (CRISPR)-Cas9 screen has been widely used to interrogate gene functions. However, the rules to design better libraries beg further refinement.ResultsWe found single guide RNA (sgRNA) outliers are characterized by higher G-nucleotide counts, especially in regions distal from the PAM motif and are associated with stronger off-target activities. Furthermore, using non-targeting sgRNAs as negative controls lead to strong bias, which can be mitigated by using sgRNAs targeting multiple ‘safe harbor’ regions. Custom-designed screens confirmed our findings and further revealed that 19 nt sgRNAs consistently gave the best signal-to-noise ratio. Collectively, our analysis motivated the design of a new genome-wide CRISPR/Cas9 screen library and uncovered some intriguing properties of the …

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May 2018 • PeerJ Computer Science

ClusterEnG: an interactive educational web resource for clustering and visualizing high-dimensional data

Mohith Manjunath, Yi Zhang, Yeonsung Kim, Steve H Yeo, Omar Sobh, Nathan Russell, Christian Followell, Colleen Bushell, Umberto Ravaioli, Jun S Song

BackgroundClustering is one of the most common techniques in data analysis and seeks to group together data points that are similar in some measure. Although there are many computer programs available for performing clustering, a single web resource that provides several state-of-the-art clustering methods, interactive visualizations and evaluation of clustering results is lacking.MethodsClusterEnG (acronym for Clustering Engine for Genomics) provides a web interface for clustering data and interactive visualizations including 3D views, data selection and zoom features. Eighteen clustering validation measures are also presented to aid the user in selecting a suitable algorithm for their dataset. ClusterEnG also aims at educating the user about the similarities and differences between various clustering algorithms and provides tutorials that demonstrate potential pitfalls of each algorithm.ConclusionsThe web resource will be particularly useful to scientists who are not conversant with computing but want to understand the structure of their data in an intuitive manner. The validation measures facilitate the process of choosing a suitable clustering algorithm among the available options. ClusterEnG is part of a bigger project called KnowEnG (Knowledge Engine for Genomics) and is available at http://education. knoweng. org/clustereng.BackgroundClustering is one of the most powerful and widely used analysis techniques for discovering structure in large datasets by grouping data points that are similar according to some measure. Several programming languages such as R (R Core Team, 2015) and Python (Pedregosa et al., 2011) offer libraries …

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Apr 2018 • Cancer research

Integrative genomic analysis predicts causative cis-regulatory mechanisms of the breast cancer–associated genetic variant rs4415084

Yi Zhang, Mohith Manjunath, Shilu Zhang, Deborah Chasman, Sushmita Roy, Jun S Song

Previous genome-wide association studies (GWAS) have identified several common genetic variants that may significantly modulate cancer susceptibility. However, the precise molecular mechanisms behind these associations remain largely unknown; it is often not clear whether discovered variants are themselves functional or merely genetically linked to other functional variants. Here, we provide an integrated method for identifying functional regulatory variants associated with cancer and their target genes by combining analyses of expression quantitative trait loci, a modified version of allele-specific expression that systematically utilizes haplotype information, transcription factor (TF)–binding preference, and epigenetic information. Application of our method to a breast cancer susceptibility region in 5p12 demonstrates that the risk allele rs4415084-T correlates with higher expression levels of the protein-coding …

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Mar 2018 • Cell systems

Genome-Scale Signatures of Gene Interaction from Compound Screens Predict Clinical Efficacy of Targeted Cancer Therapies

Peng Jiang, Winston Lee, Xujuan Li, Carl Johnson, Jun S Liu, Myles Brown, Jon Christopher Aster, X Shirley Liu

Identifying reliable drug response biomarkers is a significant challenge in cancer research. We present computational analysis of resistance (CARE), a computational method focused on targeted therapies, to infer genome-wide transcriptomic signatures of drug efficacy from cell line compound screens. CARE outputs genome-scale scores to measure how the drug target gene interacts with other genes to affect the inhibitor efficacy in the compound screens. Such statistical interactions between drug targets and other genes were not considered in previous studies but are critical in identifying predictive biomarkers. When evaluated using transcriptome data from clinical studies, CARE can predict the therapy outcome better than signatures from other computational methods and genomics experiments. Moreover, the CARE signatures for the PLX4720 BRAF inhibitor are associated with an anti-programmed death 1 …

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Mar 2018 • Oncogenesis

Expression of lncRNA MIR222HG co-transcribed from the miR-221/222 gene promoter facilitates the development of castration-resistant prostate cancer

Tong Sun, Shin-Yi Du, Joshua Armenia, Fangfang Qu, Jingyu Fan, Xiaodong Wang, Teng Fei, Kazumasa Komura, Shirley X Liu, Gwo-Shu Mary Lee, Philip W Kantoff

Mechanisms by which non-coding RNAs contribute to the progression of hormone-sensitive prostate cancer (PCa)(HSPC) to castration-resistant PCa (CRPC) remain largely unknown. We previously showed that microRNA-221/222 is up-regulated in CRPC and plays a critical role in modulating androgen receptor function during CRPC development. With further investigation, we characterized a putative promoter region located 23.3 kb upstream of the miR-221/222 gene, and this promoter is differentially activated in CRPC LNCaP-Abl cells, leading to the up-regulation of miR-221/222. Upon promoter activation, a set of polyadenylated long non-coding RNA (lncRNA) MIR222HGs was transcribed from this promoter region. Over-expression of these MIR222HGs increased androgen-independent cell growth and repressed the expression of androgen receptor-regulated dihydrotestosterone (DHT)-induced KLK3 …

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Feb 2018 • Science

A major chromatin regulator determines resistance of tumor cells to T cell–mediated killing

Deng Pan, Aya Kobayashi, Peng Jiang, Lucas Ferrari de Andrade, Rong En Tay, Adrienne M Luoma, Daphne Tsoucas, Xintao Qiu, Klothilda Lim, Prakash Rao, Henry W Long, Guo-Cheng Yuan, John Doench, Myles Brown, X Shirley Liu, Kai W Wucherpfennig

Many human cancers are resistant to immunotherapy, for reasons that are poorly understood. We used a genome-scale CRISPR-Cas9 screen to identify mechanisms of tumor cell resistance to killing by cytotoxic T cells, the central effectors of antitumor immunity. Inactivation of &gt;100 genes—including Pbrm1, Arid2, and Brd7, which encode components of the PBAF form of the SWI/SNF chromatin remodeling complex—sensitized mouse B16F10 melanoma cells to killing by T cells. Loss of PBAF function increased tumor cell sensitivity to interferon-γ, resulting in enhanced secretion of chemokines that recruit effector T cells. Treatment-resistant tumors became responsive to immunotherapy when Pbrm1 was inactivated. In many human cancers, expression of PBRM1 and ARID2 inversely correlated with expression of T cell cytotoxicity genes, and Pbrm1-deficient murine melanomas were more strongly infiltrated by …

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Feb 2018 • Cancer cell

Allele-specific chromatin recruitment and therapeutic vulnerabilities of ESR1 activating mutations

Rinath Jeselsohn, Johann S Bergholz, Matthew Pun, MacIntosh Cornwell, Weihan Liu, Agostina Nardone, Tengfei Xiao, Wei Li, Xintao Qiu, Gilles Buchwalter, Ariel Feiglin, Kayley Abell-Hart, Teng Fei, Prakash Rao, Henry Long, Nicholas Kwiatkowski, Tinghu Zhang, Nathanael Gray, Diane Melchers, Rene Houtman, X Shirley Liu, Ofir Cohen, Nikhil Wagle, Eric P Winer, Jean Zhao, Myles Brown

Estrogen receptor α (ER) ligand-binding domain (LBD) mutations are found in a substantial number of endocrine treatment-resistant metastatic ER-positive (ER+) breast cancers. We investigated the chromatin recruitment, transcriptional network, and genetic vulnerabilities in breast cancer models harboring the clinically relevant ER mutations. These mutants exhibit both ligand-independent functions that mimic estradiol-bound wild-type ER as well as allele-specific neomorphic properties that promote a pro-metastatic phenotype. Analysis of the genome-wide ER binding sites identified mutant ER unique recruitment mediating the allele-specific transcriptional program. Genetic screens identified genes that are essential for the ligand-independent growth driven by the mutants. These studies provide insights into the mechanism of endocrine therapy resistance engendered by ER mutations and potential therapeutic …

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Jan 2018 • BioRxiv

Reducing false positives in CRISPR/Cas9 screens from copy number variations

Alexander Wu, Tengfei Xiao, Teng Fei, X Shirley Liu, Wei Li

CRISPR/Cas9 knockout screens have been widely used to interrogate gene functions across a wide range of cell systems. However, the screening outcome is biased in amplified genomic regions, due to the ability of the Cas9 nuclease to induce multiple double-stranded breaks and strong DNA damage responses at these regions. We developed algorithms to correct biases associated with copy number variations (CNV), even when the CNV profiles are unknown. We demonstrated that our methods effectively reduced false positives in amplified regions while preserving signals of true positives. In addition, we developed a sliding window approach to estimate regions of high copy numbers for cases in which CNV information is not available. These copy number estimations can subsequently be used to effectively correct CNV-related biases in CRISPR screening experiments. Our approach is integrated into the existing MAGeCK/MAGeCK-VISPR analysis pipelines and provides a convenient framework to improve the precision of CRISPR screening results.

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Jan 2018 • bioRxiv

Can cancer GWAS variants modulate immune cells in the tumor microenvironment?

Yi Zhang, Mohith Manjunath, Jialu Yan, Brittany A Baur, Shilu Zhang, Sushmita Roy, Jun S Song

Genome-wide association studies (GWAS) have hitherto identified several genetic variants associated with cancer susceptibility, but the molecular functions of these risk modulators remain largely uncharacterized. Recent studies have begun to uncover the regulatory potential of non-coding GWAS SNPs by using epigenetic information in corresponding cancer cell types and matched normal tissues. However, this approach does not explore the potential effect of risk germline variants on other important cell types that constitute the microenvironment of tumor or its precursor. This paper presents evidence that the breast cancer-associated variant rs3903072 may regulate the expression of CTSW in tumor infiltrating lymphocytes. CTSW is a candidate tumor-suppressor gene, with expression highly specific to immune cells and also positively correlated with breast cancer patient survival. Integrative analyses suggest a putative causative variant in a GWAS-linked enhancer in lymphocytes that loops to the 3’ end of CTSW through three-dimensional chromatin interaction. Our work thus poses the possibility that a cancer-associated genetic variant might regulate a gene not only in the cell of cancer origin, but also in immune cells in the microenvironment, thereby modulating the immune surveillance by T lymphocytes and natural killer cells and affecting the clearing of early cancer initiating cells.

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2018 • Blood

Mass cytometry of Hodgkin lymphoma reveals a CD4 (+) exhausted T-effector and T-regulatory cell rich microenvironment

Fathima Zumla Cader, Ron CJ Schackmann, Xihao Hu, Kirsty Wienand, Robert Redd, Bjoern Chapuy, Jing Ouyang, Nicole Paul, Evisa Gjini, Mikel Lipschitz, Philippe Armand, David Wu, Jonathan R Fromm, Donna Neuberg, X Shirley Liu, Scott J Rodig, Margaret A Shipp

In classical Hodgkin lymphoma (cHL), the host anti-tumor immune response is ineffective. Hodgkin Reed-Sternberg (HRS) cells have multifaceted mechanisms to evade the immune system including 9p24. 1/CD274 (PD-L1)/PDCD1LG2 (PD-L2) genetic alterations, overexpression of the PD-1 ligands and associated T-cell exhaustion and additional structural bases of aberrant antigen presentation. The clinical success of PD-1 blockade in cHL suggests that the tumor microenvironment (TME) contains reversibly exhausted T-effectors (Teff). However, durable responses are observed in patients with β2M/MHC class I loss on HRS cells, raising the possibility of non-CD8+ T cell-mediated mechanisms of efficacy of PD-1 blockade. These observations highlight the need for a detailed analysis of the cHL TME.Using a customized time-of-flight mass cytometry (CyTOF) panel, we simultaneously assessed cell suspensions from diagnostic cHL biopsies and control reactive lymph node/tonsil (RLNT) samples. Precise phenotyping of immune cell subsets revealed salient differences between cHLs and RLNTs. The TME in cHL is CD4+ T-cell rich with frequent loss of MHC class I expression on HRS cells. In cHLs, we found concomitant expansion of Th1-polarized Teff and regulatory T cells (Treg). The cHL Th1 Tregs expressed little or no PD-1 while the Th1 Teffs were PD-1+. The differential PD-1 expression and likely functional Th1-polarized CD4+ Tregs and exhausted Teff may represent complementary mechanisms of immunosuppression in cHL.

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Nov 2017 • ACM Transactions on Graphics (TOG)

Chromablur: Rendering chromatic eye aberration improves accommodation and realism

Steven A Cholewiak, Gordon D Love, Pratul P Srinivasan, Ren Ng, Martin S Banks

Computer-graphics engineers and vision scientists want to generate images that reproduce realistic depth-dependent blur. Current rendering algorithms take into account scene geometry, aperture size, and focal distance, and they produce photorealistic imagery as with a high-quality camera. But to create immersive experiences, rendering algorithms should aim instead for perceptual realism. In so doing, they should take into account the significant optical aberrations of the human eye. We developed a method that, by incorporating some of those aberrations, yields displayed images that produce retinal images much closer to the ones that occur in natural viewing. In particular, we create displayed images taking the eye&#39;s chromatic aberration into account. This produces different chromatic effects in the retinal image for objects farther or nearer than current focus. We call the method ChromaBlur. We conducted two …

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Sep 2017 • Cancer immunology research

Clonal expansion and interrelatedness of distinct B-lineage compartments in multiple myeloma bone marrow

Leo Hansmann, Arnold Han, Livius Penter, Michaela Liedtke, Mark M Davis

Multiple myeloma is characterized by the clonal expansion of malignant plasma cells in the bone marrow. But the phenotypic diversity and the contribution of less predominant B-lineage clones to the biology of this disease have been controversial. Here, we asked whether cells bearing the dominant multiple myeloma immunoglobulin rearrangement occupy phenotypic compartments other than that of plasma cells. To accomplish this, we combined 13-parameter FACS index sorting and t-Stochastic Neighbor Embedding (t-SNE) visualization with high-throughput single-cell immunoglobulin sequencing to track selected B-lineage clones across different stages of human B-cell development. As expected, the predominant clones preferentially mapped to aberrant plasma cell compartments, albeit phenotypically altered from wild type. Interestingly, up to 1.2% of cells of the predominant clones colocalized with B-lineage …

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Sep 2017 • Journal of Vision

Rendering correct blur

Steven Cholewiak, Gordon Love, Martin Banks

Blur occurs naturally when the eye is focused at one distance and an object is present at another distance. Vision scientists and computer-graphics (CG) engineers often wish to create display images that reproduce such depth-dependent blur, but their method is incorrect for that purpose. Their method appropriately takes into account the scene geometry, pupil size, and focus distances, but does not take into account the optical aberrations of the person who will view the resulting display images. We developed a method that, by incorporating the viewer&#39;s optics, yields displayed images that produce retinal images close to those in natural viewing. Here we concentrate on the effects of longitudinal chromatic aberration. This aberration creates different chromatic effects in the retinal image for object farther vs nearer than current focus. Our method handles this correctly. Observers viewed scenes with depth-dependent …

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Jul 2017 • ACM SIGGRAPH 2017 Emerging Technologies, 1-2, 2017

Varifocal virtuality: a novel optical layout for near-eye display

Kaan Akşit, Ward Lopes, Jonghyun Kim, Josef Spjut, Anjul Patney, Peter Shirley, David Luebke, Steven A Cholewiak, Pratul Srinivasan, Ren Ng, Martin S Banks, Gordon D Love

Augmented reality (AR) has recently gained momentum in the form of a variety of available optical see-through near-eye displays (NEDs) such as the Meta 2and the Microsoft Hololens. These devices are a big step forward towards Sutherland&#39;s vision of an ultimate display [Sutherland 1968]. The device we demonstrate attempts to deal with the main limitations of current devices. First, the graphics images are at a constant virtual distance for the eyes&#39; accommodation mechanism, while the vergence of the two eyes working in concert places the virtual object (s) at a distance other than the accommodation distance. This vergence-accommodation conflict is one of the main problems in many AR and VR systems [Kress and Starner 2013]. The second limitation is achieving a wide FOV with compact optics. Cakmakci et al.[2006] contend that achieving a wide field-of-view (FOV) is the major optical design challenge in AR …

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Jun 2017 • 3D Image Acquisition and Display: Technology, Perception and Applications …, 2017

ChromaBlur: Rendering Chromatic Eye Aberration Improves Accommodation and Realism in HMDs

Martin S Banks, Steven A Cholewiak, Gordon D Love, Pratul Srinivasan, Ren Ng

We developed a rendering method that takes into account the eye’s chromatic aberration. Accommodation is driven much more accurately with this method than with conventional methods. Perceived realism is also improved.

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