Flow cytometry data mining by cytoChain identifies determinants of exhaustion and stemness in TCR‐engineered T cells

Abstract

The phenotype of infused cells is a major determinant of Adoptive T‐cell therapy (ACT) efficacy. Yet, the difficulty in deciphering multiparametric cytometry data limited the fine characterization of cellular products. To allow the analysis of dynamic and complex flow cytometry samples, we developed cytoChain, a novel dataset mining tool and a new analytical workflow. CytoChain was challenged to compare state‐of‐the‐art and innovative culture conditions to generate stem‐like memory cells (TSCM) suitable for ACT. Noticeably, the combination of IL‐7/15 and superoxides scavenging sustained the emergence of a previously unidentified nonexhausted Fit‐TSCM signature, overlooked by manual gating and endowed with superior expansion potential. CytoChain proficiently traced back this population in independent datasets, and in T‐cell receptor engineered lymphocytes. CytoChain flexibility and function were then further validated on a published dataset from circulating T cells in COVID‐19 patients. Collectively, our results support the use of cytoChain to identify novel, functionally critical immunophenotypes for ACT and patients immunomonitoring.

CAR T cell manufacturing from naive/stem memory T lymphocytes enhances antitumor responses while curtailing cytokine release syndrome

“…To answer this question, we performed the same experiment as described in Figure 2A , but with the aim of deepening the phenotypic characterization of CAR T cells after the first response, i.e., at day 14 after CAR T cell infusion. To this aim, we sought to employ an unsupervised approach based on the Barnes-Hut stochastic neighbor embedding (BH-SNE) dimensionality reduction algorithm for data analysis ( 36 – 38 ).…”

Immunotherapeutic Potential of T Memory Stem Cells

“…What's more, CAR-T/IL-15 cells exhibited reduced expression of exhaustion markers, higher anti-apoptotic properties, and increased proliferative capacity when it was attacked by antigens (57). The combined use of IL-7 and IL-15 can preserve the T SCM phenotype and enhance the effectiveness of CAR-T cells (11,29,(58)(59)(60)(61). IL-21 was critical for the long-term maintenance and functionality of (67).…”

Workflow for high-dimensional flow cytometry analysis of T cells from tumor metastases

“…We challenged the accuracy and sensitivity of our workflow by verifying whether also less characterized tumor-infiltrating unconventional T-cell populations could be identified by unsupervised computational analysis, in addition to classical analysis approaches. Unsupervised analysis of the previously described CRC-LM infiltrating T-cell data set ( Fig 5 ) was carried out by cytoChain, a recently published web application for HD flow cytometry data mining ( Manfredi et al, 2021 ). The cytoChain modular pipeline includes pre-analytical steps to correct flow cytometer fluctuations and multidimensional data scattering; evaluation of the appropriate HD analysis according to the specific data set qualities; and quantitative analysis to identify clusters of cells sharing similar phenotypes with an exhaustive graphical output.…”

“…To further dissect the data set, we applied the FlowSOM algorithm ( Van Gassen et al, 2015 ), which could not selectively assign iNKT cells to any cluster or meta-cluster even though they were tightly grouped on the t-SNE map ( Fig S16A and B ). To overcome these problems, we implemented our recently described cytoChain application ( Manfredi et al, 2021 ) with FastPhenoGraph (FastPG) algorithm ( Fig S16C ) ( Bodenheimer et al, 2020 Preprint ), which computed 28 clusters that were superimposed on the t-SNE map, allowing both the visualization of their distribution within the data set ( Fig 8B ) and of their phenotype by a heat-map generated based on the fluorescence intensity associated to each expressed marker ( Fig 8C ). For instance, a specific phenotype signature was found to be enriched among CD8 T cells for clusters 21, 17, 24, 13, 27, and 23 that expressed both tissue residence (CD69-CD103) and activation/exhaustion markers to a variable extent (i.e., CD39, CD95, TIGIT, 2B4, PD1, HLA-DR, ICOS, and GITR).…”

“…In the present work, we describe a novel methodological workflow for HD flow cytometric analysis of tumor-infiltrating T cells, including conventional effector, Treg, T FH , and unconventional T-cell subsets from CRC-derived liver metastases (CRC-LM), encompassing: (1) application of a recently developed cell-based calibration protocol for optimizing the flow cytometer setting; (2) design of a novel 26-color based T-cell panel sampling both conventional and unconventional T-cell subsets; (3) implementation of a protocol to isolate single cell suspensions from surgically resected CRC and CRC-LM. The combination of these accurate experimental procedures with the data analysis by cytoChain ( Manfredi et al, 2021 ), a recently described in silico workflow for flow high-dimensional analysis, offers a reliable tool for the characterization of the liver metastatic T-cell landscape.…”