Identification of new cytokine combinations for antigen-specific T-cell therapy products via a high-throughput multi-parameter assay |
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| Authors: | Christopher A Lazarski Anushree A Datar Emily K Reynolds Michael D Keller Catherine M Bollard Patrick J Hanley |
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| Institution: | 1. Center for Cancer and Immunology Research, Children''s National Hospital, Washington, DC, USA;2. Division of Allergy and Immunology, Children''s National Hospital, Washington, DC, USA;3. Division of Blood and Marrow Transplantation, Children''s National Hospital, Washington, DC, USA;4. The George Washington University Cancer Center, Washington, DC, USA;1. Vinmec Research Institute of Stem Cell and Gene Technology, Vinmec Healthcare System, Hanoi, Vietnam;2. Vinmec HiTech Center, Vinmec Healthcare System, Hanoi, Vietnam;3. College of Health Science, VinUniversity, Hanoi, Vietnam;1. Department of Chemistry and Biotechnology, Faculty of Science, Engineering and Technology, Swinburne University of Technology, Hawthorn, Australia;2. Regenerative Medicine and Stem Cell Laboratory, Department of Biomedical Engineering, Indian Institute of Technology Hyderabad, Medak, India;3. ARC Training Centre in Surface Engineering for Advanced Materials, School of Engineering, Swinburne University of Technology, Hawthorn, Australia;1. Department of Spine Surgery, The Third Affiliated Hospital of Sun Yat-sen University, Guangdong Provincial Center for Engineering and Technology Research of Minimally Invasive Spine Surgery, Guangdong Provincial Center for Quality Control of Minimally Invasive Spine Surgery, Guangzhou, People''s Republic of China;2. Cell-Gene Therapy Translational Medicine Research Center, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, People''s Republic of China;1. University of California San Diego Moores Cancer Center, La Jolla, California, USA;2. PersImmune, Inc, San Diego, California, USA;3. University of California San Diego Apheresis Program and Division of Nephrology, La Jolla, California, USA;1. Department of Hematology, Leiden University Medical Center, Leiden, The Netherlands;2. Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands;3. Center for Clinical Transfusion Research, Sanquin Research, Leiden, The Netherlands;4. Department of Hematology, HagaZiekenhuis, The Hague, The Netherlands |
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| Abstract: | 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. Ten-day cultures were tested by 13-color flow cytometry to evaluate viable cell count, lymphocyte phenotype, memory markers and interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) 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+ CCR7– effector memory (CD45RO+ CCR7– CD3+), central memory (CD45RO+ CCR7+ CD3+), terminal effector (CD45RO– CCR7– CD3+), and naive (CD45RO– CCR7+ CD3+). T cells were the preponderant cells (76.8% and 72.3% in IL-15/IL-6 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 IFNγ (7.6% producing both TNFα and IFNγ) and 18.5% of CD3+ cells grown in IL-4/IL-7 producing IFNγ (9% producing both TNFα and IFNγ). This study shows the utility of this single-plate assay to rapidly identify optimal growth conditions for VST manufacture using only 107 PBMCs. |
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