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

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 good manufacturing practice ( 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 analysing 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 analysed using a 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 interleukin (IL)‐2, tumour necrosis factor (TNF)‐α and interferon (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.