Cytometric analysis of T cell phenotype using cytokine profiling for improved manufacturing of an EBV-specific T cell therapy

Rachel Cooper, Aleksandra Kowalczuk, Gwen Wilkie, Mark Vickers, Marc Turner, John Campbell, Alasdair Fraser* (Corresponding Author)

*Corresponding author for this work

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Adoptive immunotherapy using Epstein-Barr Virus (EBV)-specific T cells is a potentially curative treatment for patients with EBV-related malignancies where other clinical options have proved ineffective. We describe improved GMP-compliant culture and analysis processes for conventional lymphoblastoid cell line (LCL)-driven EBV-specific T cell manufacture, and describe an improved phenotyping approach for analyzing T cell products. We optimized the current LCL-mediated clinical manufacture of EBV-specific T cells to establish an improved process using xenoprotein-free GMP-compliant reagents throughout, and compared resulting products with our previous banked T cell clinical therapy. We assessed effects of changes to LCL: T cell ratio in T cell expansion, and developed a robust flow cytometric marker panel covering T cell memory, activation, differentiation and intracellular cytokine release to characterize T cells more effectively. These data were analyzed using t-Stochastic Neighbour Embedding (t-SNE) algorithm. The optimized GMP-compliant process resulted in reduced cell processing time and improved retention and expansion of central memory T cells. Multi-parameter flow cytometry determined the optimal protocol for LCL stimulation and expansion of T cells and demonstrated that cytokine profiling using IL-2, TNF-α and IFN-γ was able to determine the differentiation status of T cells throughout culture and in the final product. We show that fully GMP-compliant closed-process culture of LCL-mediated EBV-specific T cells is feasible and profiling of T cells through cytokine expression gives improved characterization of start material, in-process culture conditions and final product. Visualization of the complex multi-parameter flow cytometric data can be simplified using t-SNE analysis.
Original languageEnglish
Article number68-81
Number of pages14
JournalClinical and Experimental Immunology
Issue number1
Early online date14 Jul 2021
Publication statusPublished - 31 Oct 2021

Bibliographical note

We gratefully acknowledge the voluntary donations of peripheral blood and leukapheresis samples used in this
study. This study was supported by the staff of the Scottish National Blood Transfusion Service (SNBTS)
Clinical Apheresis Unit, Aberdeen and medical colleagues at the New Zealand Blood Transfusion Service for collection of the donor leukapheresis material for the second iteration of the EBV-T cell bank. This work was supported by a Wellcome Trust Translational Award. The ongoing operation of the UK EBV-specific T cell bank is supported by cost-recovery fees and supported by SNBTS.
RSC and AK were responsible for the acquisition and analysis of data, and RSC wrote the principal manuscript; GW and MV were involved in supply of material and interpretation of data; MLT, JDMC and ARF were responsible for study conception and design, data analysis and interpretation, and revision of the final manuscript.


  • cancer
  • cell differentiation
  • cytotoxic T-cells
  • T-cells
  • viral


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