Faculty Bibliography
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94
Immune monitoring for immuno-oncology applications [Meeting Abstract]
The immunotherapy revolution has spurred the development of many new drugs and drug regimens for patient treatment. A key challenge is to identify the factors that drive patient toxicities and responses to treatment, with a particularly acute need for predictive biomarkers that can discriminate patients destined to respond and fail treatment. Technologies to interrogate immune cells are now readily available, but important gaps remain in their application, which limit the full realization of the promise of precision oncology. High parameter flow cytometry is a gold-standard but is limited by difficulty of panel design and lack of standardization. We present Color Wheel, a panel design tool, which builds optimized antibody panels based on the user's instrument. These optimized panels have been manufactured in a dried, ready to use format to drive workflow and assay standardization. Initial results demonstrate excellent concordance between the dried and liquid versions of the high parameter multicolor panel(s). Molecular cytometry represents an exciting new approach to high dimensional immune analysis because it can measure at least 102 proteins and 400 mRNA targets simultaneously per cell. An important application gap for this technology is lack of data indicating the sequencing depth needed for adequate resolution. Here, we present results from a molecular cytometry experiment sequenced deeply, and then bioinformatically subsampled at different levels to identify the minimum level of sequencing needed for clear identification of cells, which can serve as a reference guide for users to save time and cost in their experiments. We also present preliminary data generated using dried version of molecular cytometry panel(s)
EMBASE:633105648
ISSN: 1550-6606
CID: 4638892
Deep characterization of in vitro chronically stimulated T cells via single-cell multiomic analysis [Meeting Abstract]
To generate enough cells for viable adoptive cell therapy, cells must be robustly stimulated, which raises the risk of inducing T-cell exhaustion and reducing therapeutic efficacy. To comprehensively characterize in vitro chronically expanded human T cells, we performed single-cell multiomic analysis using BD AbSeq and BD RhapsodyTM Single-Cell Analysis system for simultaneous measurement of the expression of 38 proteins and 399 genes. Comprehensive immunophenotypic and transcriptomic analysis at day 0 enabled a refined characterization of T-cell maturational states. T-cell activation induced by anti-CD3/CD28 beads and recombinant human IL-2 led to downregulation of naive-associated markers and upregulation of effector molecules, proliferation regulators, co-inhibitory and co-stimulatory receptors. Our deep kinetic analysis further revealed clusters of proteins and genes identifying unique states of activation defined by markers temporarily expressed upon 3 days of stimulation, markers constitutively expressed throughout chronic activation, and markers uniquely up-regulated upon 14 days of stimulation. These data indicate heterogeneity and plasticity of chronically stimulated T cells in response to different kinetics of activation. We demonstrate the power of a single-cell multiomic approach to comprehensively characterize T cells and to precisely monitor changes in differentiation, activation and exhaustion signatures in response to different activation protocols. For Research Use Only. Not for use in diagnostic or therapeutic procedures. BD, the BD Logo, and Rhapsody are trademarks of Becton, Dickinson and Company or its affiliates
EMBASE:633105356
ISSN: 1550-6606
CID: 4638902
Molecular cytometry identifies a wide range of translationally relevant markers in tumor and peripheral immune cells of lung cancer patients [Meeting Abstract]
Many immunotherapy drugs, used singly or in combination, are emerging. To realize precision oncology, and better design clinical trials, in-depth immune profiling is key. Many new technologies are available; the most promising simultaneously analyzes >102 proteins and 400 mRNA cell-by-cell (molecular cytometry). Using this technology, we deeply profiled TIL, from freshly resected lung tissue, and PBMC collected at surgery (n=10). Antibody staining was robust, with all canonical cell populations at expected frequency. We identified markers uniquely upregulated in TIL, including CXCR6, CD39, CD26, CD69, CD103, and RGS. We also asked what markers were uniquely enriched in PD1-TIL, in order to find other drug targets for patients who fail Nivolumab. We found many molecules upregulated in PD1-TIL, including CD326, CD98, LGALS3, TIM3, CD54, CD235ab, CXCL8, CD141, and CD117. We further identified precise combinations of these markers that inform design of combination immunotherapy. We also characterized immune landscapes in metastatic disease vs. localized adenocarcinoma, finding drug targets and tumor-immune interactions are different across these settings. For example, T-cell activation markers are downregulated in metastatic tissue, e.g., HLADR (p = 1.5E-16), CD69 (p=2.9e-10), and CD38 (p=3.2e-16). CD103 was also highly downregulated (p=1.9E-14). Notably, in metastatic disease various myeloid proteins are elevated, including CD206 (p=0.002), CD32 (p=0.02), and CD61 (p=0.006). We also characterized the degree of immune exhaustion, using ratios of ZBED2:LGALS in cells. In summary, we demonstrate the utility of molecular cytometry for providing unique (and translationally-relevant) insight into lung cancer.
EMBASE:633105196
ISSN: 1550-6606
CID: 4638912
Deep characterization of in vitro chronically stimulated T cells through single-cell multiomic analysis [Meeting Abstract]
A key step in the clinical production of CAR T cells is the expansion of engineered T cells. To generate enough cells for a therapeutic product, cells must be robustly stimulated, which raises the risk of inducing T-cell exhaustion and reducing therapeutic efficacy. As protocols for T-cell expansion are being developed to optimize CAR T cell yield, function, and persistence, fundamental questions about the impact of in vitro manipulation on T-cell identity are important to answer. Namely: 1) What types of cells are generated during chronic stimulation? 2) How many unique cell states can be defined during chronic stimulation? We sought to answer these fundamental questions by performing single-cell multiomic analysis to simultaneously measure expression of 38 proteins and 399 genes in human T cells expanded in vitro. This approach allowed us to study-with unprecedented depth-how T cells change over the course of chronic stimulation. Human PBMCs from three healthy donors were continuously stimulated for 14 days in the presence of CD3/CD28 antibody-coated beads and recombinant human IL-2. This model system was developed to resemble culture conditions that may be used for CAR T cell expansion. Cells were collected at different time points (day 0, 3, 7, and 14) prior to downstream analysis. Comprehensive immunophenotypic and transcriptomic analysis at day 0 enabled a refined characterization of T-cell maturational states (from naive to TEMRA cells) and the identification of a donor-specific subset of terminally differentiated T-cells that would have been otherwise overlooked using canonical cell classification schema. As expected, T-cell activation induced downregulation of naive-associated markers and upregulation of effector molecules, proliferation regulators, coinhibitory and costimulatory receptors. Our deep kinetic analysis further revealed clusters of proteins and genes identifying unique states of activation defined by markers temporarily expressed upon 3 days of stimulation (PD-1, CD69, LTA), markers constitutively expressed throughout chronic activation (CD25, GITR, LGALS1), and markers uniquely upregulated upon 14 days of stimulation (CD39, ENTPD1, TNFSF10). Notably, different ratios of cells expressing activation or exhaustion markers were measured at each time point. These data indicate high heterogeneity and plasticity of chronically stimulated T cells in response to different kinetics of activation. In this study, we demonstrate the power of a single-cell multiomic approach to comprehensively characterize T cells and to precisely monitor changes in differentiation, activation, and exhaustion signatures in response to different activation protocols
EMBASE:631313299
ISSN: 2326-6074
CID: 4381052
The next frontier in single cell analysis: multimodal studies and clinical translation [Editorial]
PMID: 31591632
ISSN: 1473-0189
CID: 4129492
Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition)
These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion.
PMID: 31633216
ISSN: 1521-4141
CID: 4146932
Cell phenotypes associated with response and toxicity defined by high resolution flow cytometry in melanoma patients receiving checkpoint inhibition [Meeting Abstract]
Background: Peripheral blood T cell and myeloid-derived suppressor cells (MDSC), as well as myeloid and macrophage subsets, have been associated with a poor clinical outcome in a variety of cancers. We analyzed circulating cells from patients (pts) that received either nivolumab (NIVO) then ipilimumab (IPI) (cohort A, 16 pts) or IPI then NIVO (cohort B, 17 pts) in a randomized clinical trial to determine if peripheral blood phenotypes were associated either at baseline or on treatment with outcome. Method(s): Frozen peripheral blood mononuclear cells (PBMC) from the ChekMate 064 study were assessed at baseline and on treatment at week 13 for circulating cell subsets by 28-color, high-dimensional flow cytometry with CytoBrute, a rapid computational platform that performs high-parameter Boolean analysis. Correlations with response and survival as well as toxicity were evaluated using the machine learning algorithm ElasticNet. Result(s): In cohort B pts the frequency of resting Ki67-, long-lived memory CD45+/ CD45RO+/CD127+ T cells was reduced (p=0.005), and dividing Ki-67+ CD4+/ CD45RO+/CD95+ T cells susceptible to apoptosis were increased after IPI (p=0.007), but were associated at baseline with a poor outcome with cohort A (p=0.0002). Subsets of myeloid cells that were CD66b+/CD33+/41-BB+/CD86+ at baseline were associated with survival in cohort B (p=0.0006). A macrophage subset that was PD-L2+/CD163+/41-BBL+/CD40+ was associated with survival for cohort A (p=0.0001). Additional phenotypes were associated with grade 1 compared with grades 2-4 toxicity that differentiated side effects from either IPI or NIVO, and other phenotypes distinguished normals and pts (AUC=0.96). Conclusion(s): A circulating CD4+/CD45RO+/CD95+ proliferating memory T cell phenotype signature is augmented after IPI and is associated at baseline with poor survival with NIVO in CheckMate 064. We discriminated pts and healthy controls with great specificity and sensitivity at baseline, and demonstrated new phenotypes associated with immune-related toxicity. Peripheral blood immune monitoring may be of value in selecting melanoma pts to be treated with immunotherapy
EMBASE:630607281
ISSN: 1569-8041
CID: 4286042
High-Parameter Single-Cell Analysis
Thousands of transcripts and proteins confer function and discriminate cell types in the body. Using high-parameter technologies, we can now measure many of these markers at once, and multiple platforms are now capable of analysis on a cell-by-cell basis. Three high-parameter single-cell technologies have particular potential for discovering new biomarkers, revealing disease mechanisms, and increasing our fundamental understanding of cell biology. We review these three platforms (high-parameter flow cytometry, mass cytometry, and a new class of technologies called integrated molecular cytometry platforms) in this article. We describe the underlying hardware and instrumentation, the reagents involved, and the limitations and advantages of each platform. We also highlight the emerging field of high-parameter single-cell data analysis, providing an accessible overview of the data analysis process and choice of tools. Expected final online publication date for the Annual Review of Analytical Chemistry Volume 12 is June 12, 2019. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
PMID: 30699035
ISSN: 1936-1335
CID: 3626752
ISAC 34th International Congress Vancouver, Canada June 22 - 26, 2019 [Editorial]
PMID: 31087529
ISSN: 1552-4930
CID: 3919612
Novel Multi-Parameter Flow Cytometry Approaches and Data Analysis Tools for the Evaluation and Detection of Leukemia Stem Cells [Meeting Abstract]
ISI:000478081102402
ISSN: 0023-6837
CID: 4047702
