June 22 - 26, 2019
Vancouver Convention Center
Visit our booth at CYTO 2019 to learn about our innovative platforms and products.
- Intellicyt iQue® Screener PLUS - platform integrates a flow based instrument with enabling and easy to use software and reagents designed to address key challenges across the suspension-cell screening workflow.
- arium® - platform integrates a flow based instrument with enabling and easy to use software and reagents designed to address key challenges across the suspension-cell screening workflowour laboratory-grade water purification systems feature an inspiring, application-oriented design. They enable you to perform your workflows faster and more reliably, simplifying your daily lab work while ensuring cost-efficient operation over the long term.
- Picus® NxT Electronic Pipette - comfortable to hold and reduces the risk of strain injuries, conforms to the strictest lab regulations, speeds up your work, and helps you to achieve accurate results time after time.
At the show, please visit our poster presentation.
Title: Screening Ex Vivo Conditions that Increase Memory T Cell Frequency using High-Throughput Flow Cytometry and an Optimized Multiplexed Assay
Program Number: 496
Poster Board Number: B358
Authors: Andrea Gomez-Donart, Zhaoping Liu & John O’Rourke.
Essen Bioscience Inc., Part of the Sartorius Group. 5700 Pasadena Ave. NE, Albuquerque, NM 87113
A critical process in bio-manufacturing of adoptive cell therapies such as chimeric antigen receptor (CAR) T and tumor infiltrating lymphocyte (TIL) therapies is the ex vivo expansion of T cells. Recent clinical studies show a correlation between Ex Vivo expansion and persistence of infused T cells and patient outcomes. Additional studies show that a subset of functional memory T cells including T memory stem cells (Tscm), central memory T cells (Tcm) and other less differentiated T cell subsets are responsible for long-term anti-tumor responses. This suggests that ex vivo T cell expansion protocols generating higher percentages of Tscm and Tcm in the total cell product are critical to significant clinical improvements in adoptive cell therapies.
To address the need to monitor T cell phenotype and function for improved ex vivo expansion protocols where profiling of memory subsets is crucial, we developed a robust, high content T memory cell and cytokine profiling assay. This miniaturized assay uses high throughput flow cytometry to measure memory cell phenotype, cell viability and effector cytokine release in the same sample well of a 96- or 384-well microtiter plate. The optimized antibody panel includes markers to identify T cells (CD3, CD4, and CD8), markers to discriminate between naive, memory, and effector subsets (CD45RA, CD45RO, CD62L, CD95 and CD27). In addition, secreted cytokine quantitation (IFNγ and IL-10) is performed simultaneously in the same cell/bead-based assay.
Proof-of-concept screening studies were performed with human peripheral blood mononuclear cells (PBMC) from 3 donors activated with anti-CD3/anti-CD28 coated beads and supplemented with 14 different combinations of cytokines including: IL-4, IL-6, IL-7, IL-15, IL-21 and IFNβ. On Days 2 through 8 post stimulation, a small aliquot of cells and supernatant from each culture well was transferred to an assay plate and assessed for phenotype and function using the T cell memory assay. Data were acquired using the Intellicyt iQue Screener PLUS® VBR high throughput flow cytometer and analyzed using the integrated ForeCyt® software package. Expansion of patient derived T cells showed variation in phenotype and function between donors. In spite of these differences, T-cell expansion in cytokine cocktails containing IFNβ promoted the frequency of T central memory cells whereas cocktails containing IL-21 increased the frequency of T stem cell-like memory cells. Furthermore, the secretion of IL-10 correlated with the frequency of T central memory cells. These data show the T cell memory assay combined with high throughput flow cytometry is a powerful tool to rapidly screen for ex vivo culture conditions and media supplements leading to preferential expansion of desired T cell memory subsets. Besides their use in optimizing cell manufacturing protocols, these tools can be easily applied to the profiling of other biologics and drugs where the monitoring of T cell memory subsets is required.