Timour Baslan, Research Fellow at Memorial Sloan Kettering Cancer Center

Single-cell genomics and transcriptomics are revolutionizing the study of organismal genetics and biology in homeostasis and disease. Initially highly dependent on processing of single-cells isolated via flow cytometry for subsequent sequencing, in recent past, microfluidic approaches have dominated the field, often at the perceived expense of flow cytometry. Here, I will present arguments for an essential role for flow cytometry in the continued evolution and application of single-cell sequencing approaches with examples from ongoing research as well as published literature.

Leila Eslamizar, Scientist, Nonclinical Drug Safety at Boehringer Ingelheim

Cellular immunophenotyping by flow cytometry is a common tool to evaluate complex immunological processes. Many reagent companies provide a variety of labeled antibodies and fluorescent-based kits to help scientists to achieve such goals. In order to design complex immunophenotyping assays minimum technical problems, scientists need to consider a few important concepts such as the properties of dyes, target receptor density, and the level of detection sensitivity when designing complex panels. In this interactive discussion, some of these important factors will be illustrated with practical examples

Jared Burks, Associate Professor & Co-Director Flow Cytometry & Cell Imaging Core Facility at The University of Texas MD Anderson Cancer Center

When contemplating multiplex studies what can be gained and how should the study be structured? What features are important to the study and ultimately why perform the time consuming, costly, and more challenging experiment? There are multiple ways to answer these questions, but my answers may surprise. Most of my perspective derives from what we have learned from other technologies and how we extrapolated those studies. My goal is to inform and help researchers determine their best options from suspension to imaging studies using Mass Cytometry. Ultimately, it all comes down the correct analytical tool and learning what to look for in the clutter. … just imagine

Bei Wang, Senior Staff Scientist at Regeneron Pharmaceuticals

Most patients with cancer do not develop durable antitumor responses after PD-1 or PD-L1 checkpoint inhibition monotherapy because of an ephemeral reversal of T cell dysfunction and failure to promote long-lasting immunological T cell memory. Activating costimulatory pathways to induce stronger T cell activation may improve the efficacy of checkpoint inhibition and lead to durable antitumor responses. We performed single-cell RNA sequencing and high dimensional flow cytometry of tumor-infiltrating CD8+ T cells in mice receiving both PD-1 and GITR antibodies and found that this combination synergistically enhanced the effector function of expanded CD8+ T cells by restoring the balance of key homeostatic regulators CD226 and TIGIT, leading to a robust survival benefit. Combination therapy decreased CD8+ T cell dysfunction and induced a highly proliferative precursor effector memory T cell phenotype in a CD226-dependent manner. PD-1 inhibition rescued CD226 activity by preventing PD-1–Src homology region 2 (SHP2) dephosphophorylation of the CD226 intracellular domain, whereas GITR agonism decreased TIGIT expression. Unmasking the molecular pathways driving durable antitumor responses will be essential to the development of rational approaches to optimizing cancer immunotherapy.

Pratip K. Chattapadhyay, Associate Professor (Pathology) & Director, Precision Immunology Incubator, Isaac and Laura Perlmutter Cancer Center, NYU Langone Medical Center

The ability to simultaneously query protein and mRNA has emerged only recently, with development of CITE-seq, REAP-seq, and Ab-seq platforms. Each of these “molecular cytometry” technologies rely on antibodies conjugated to oligonucleotide tags, followed by capture of stained cells for single cell RNA-seq. Molecular cytometry has important advantages over flow cytometry - chiefly, a limitless number of parameters and measurements of gene expression. We used the newest molecular cytometry technique (Ab-seq, BD Biosciences) to better understand performance and utility of this class of technologies. We also present molecular cytometry data from our recent work on an in vitro model of T-cell activation/exhaustion, which likely has direct relevance to CAR T-cell therapy, and from the study of tumor-infiltrating lymphocytes (TIL) in lung cancer tissue.

Herve Luche, Scientific Director, Immunophenotyping Module at CIPHE at Inserm

Panel design is a complex and time-consuming task in multi-color flow cytometry. This complexity increases exponentially according to the number of colors/reagents used simultaneously.

To obtain an equilibrated panel with an optimal resolution of all cell populations, one of the main rules is to target high expressed molecules with dim fluorochromes and low expressed molecules with bright reagents. Fluorochrome brighteness depends on cytometer configuration and can be determined using stain index. However, antigen density is often inferred from results of fluorescence intensity found in the literature or available on company websites. Attribution of a marker to a given class of antigen expression relies on the initial knowledge/assumption of scientists for a given population. Instrument configuration used to generate these results is rarely available and hampers the accurate translation of this knowledge to a given study. Likewise, it is hard to extensively know the co-expression of all cell surface molecules in inflammatory conditions and there is currently no database available with such information on multiple immune cell types.
To rationalize panel design, the immunophenotyping module of CIPHE, in partnership with BD Biosciences, has investigated the DYnamic of Antigen DEnsity in the Mouse (DYADEM). 450 surface molecules were quantified on 16 populations of murine splenocytes, at steady state or under inflammatory conditions (poly-IC or anti-CD3e trigger). Measurements were done in the PE channel of a BD LSRII Fortessa equipped with a 561nm, 100 mW laser line in order to improve the detection of dimly expressed proteins. MFI was related to a number of molecules was using BD Quantibrite bead standards and expressed in Antibody bound per Cell (AbC) units. For all 450 molecules, quantification were performed in triplicate on independent mice for all inflammatory conditions. This dataset represents almost 5000 FCS files acquired in a standardized manner. To reduce variation of the results brought by traditional supervised analysis methodologies, analysis was not performed manually. A semi-automatic, unsupervised analysis was set up in R and interfaced to biologists using R-shiny. Compensation and scales around zero were verified on each FCS files by two biologists before Scaffold analysis. Clusters generated by the CLARA algorithm were mapped onto the same reference map of landmarks that were manually gated population. Population annotation of each cluster was transferred to single cells within the FCS file: a population index was created as a new parameter in order to ease the cross-validation of automated annotation in commercial softwares.
Details about the assay setup and data analysis pipeline will be presented. This extensive dataset of antigen density measurements in the mouse should be very instrumental for faster definition of high content panels. It should help the community of fundamental immunologists working on this model to build more robust panels for studies of inflammation. Comparing these results with similar projects run in human may help to find more robust phenotypic keys to ease initiation of translational studies.