Identification of new cytokine combinations for antigen-specific T-cell therapy products via a high-throughput multi-parameter assay

Infusion of viral-specific T cells (VSTs) is an effective treatment for viral infection after stem cell transplant. Current manufacturing approaches are rapid, but growth conditions can still be further improved. To optimize VST cell products, the authors designed a high-throughput flow cytometry-based assay using 40 cytokine combinations in a 96-well plate to fully characterize T-cell viability, function, growth and differentiation. Peripheral blood mononuclear cells (PBMCs) from six consenting donors were seeded at 100 000 cells per well with pools of cytomegalovirus peptides from IE1 and pp65 and combinations of IL-15, IL-6, IL-21, interferon alpha, IL-12, IL-18, IL-4 and IL-7. Tenday cultures were tested by 13-color flow cytometry to evaluate viable cell count, lymphocyte phenotype, memory markers and interferon gamma (IFN gamma) and tumor necrosis factor alpha (TNF alpha) expression. Combinations of IL-15/ IL-6 and IL-4/IL-7 were optimal for the expansion of viral-specific CD3+ T cells, (18-fold and 14-fold, respectively, compared with unstimulated controls). CD8+ T cells expanded 24-fold in IL-15/IL-6 and 9-fold in IL-4/IL-7 cultures (P < 0.0001). CD4+ T cells expanded 27-fold in IL-4/IL-7 and 15-fold in IL-15/IL-6 (P < 0.0001). CD45RO+ CCR7effector memory (CD45RO+ CCR7CD3+), central memory (CD45RO+ CCR7+ CD3+), terminal effector (CD45RO CCR7CD3+), and naive (CD45ROCCR7+ CD3+). T cells were the preponderant cells (76.8% and 72.3% in IL-15/IL6 and IL-15/IL-7 cultures, respectively). Cells cultured in both cytokine conditions were potent, with 19.4% of CD3+ cells cultured in IL-15/IL-6 producing IFNg (7.6% producing both TNFa and IFNg) and 18.5% of CD3+ cells grown in IL-4/IL-7 producing IFNg (9% producing both TNF alpha and IFN gamma). This study shows the utility of this single-plate assay to rapidly identify optimal growth conditions for VST manufacture using only 107 PBMCs. (C) 2020 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.