Abstract 1209: Screening ex vivo conditions that increase memory T cell frequency using high throughput flow cytometry and an optimized multiplexed assay
Authors: Liu Zhaoping , Donart-Gomez Andrea , O'Rourke John 
Published: August 2019

Abstract
A critical process in bio-manufacturing of adoptive cell therapies such 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 in 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) and central memory T cells (Tcm) are responsible for the majority of in vivo expansion and persistence leading to increased anti-tumor responses. This suggests that ex vivo protocols generating higher percentages of Tscm and Tcm in the total cell product will lead to significant clinical improvements.
We developed a robust, high-content T cell memory assay to address monitoring requirements for T cell phenotype and function for improved ex vivo expansion protocols and other studies where profiling of memory subsets is crucial. This miniaturized assay uses high throughput flow cytometry to measure 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, effector subsets (CD45RA, CD45RO, CD62L, and CD95); and a long-term survival marker (CD27). In addition, secreted cytokine quantitation (INFγ and IL-10) is performed simultaneously with the phenotypic and cell health measurements using a bead-based assay.
Proof of concept studies were performed by stimulating peripheral blood mononuclear cells (PBMC) from multiple donors using anti-CD3/anti-CD28 coated beads. Activated T cells were expanded by culturing in serum-free media supplemented with different combinations and concentrations of IL-4, IL-7, IL-15, and IL-21 in a 96-well plate format. On Days 3 through 7 post stimulation, a small aliquot 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. These data showed that the various cytokine combinations had a distinct effect on promoting T cell memory frequency, especially the Tscm cell subset, as well as effector cytokine secretion in a temporal and concentration specific manner.
These data show the T cell memory assay, combined with high throughput flow cytometry, is a powerful tool to rapidly screen for cytokine combinations that increase the frequency of T-memory cells during ex vivo expansion of T-cell products. 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.
Citation Format: Zhaoping Liu, Andrea Donart-Gomez, John O'Rourke. Screening ex vivo conditions that increase memory T cell frequency using high throughput flow cytometry and an optimized multiplexed assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1209.