Lower bead to T cell ratios support the generation of human Th17 cells with stem cell memory properties and enhanced antitumor activity
Lillian R. Neal1, Logan W. Huff1, Michelle H. Nelson1, Megan M. Wyatt1, Stefanie R. Bailey1, Jacob S. Bowers1, Sreenath Kundimi1, Shikhar Mehrotra1, Juan C. Varela1, Chrystal M. Paulos1
1Medical University of South Carolina, Charleston, SC, USA
Recent reports demonstrated that tumor-reactive Th17 cells regressed large human tumors when infused into mice. Yet the ideal amount of activation signal strength to expand Th17 cells with durable memory and enhanced functionality is unknown. To address this question, human Th17 cells were activated with decreasing concentration of CD3/CD28 or CD3/ICOS-paramagnetic beads used in culture for activation [high, 3 beads: 1 T cell; medium, 1:10; low, 1:100]. We discovered that CAR+Th17 cell cultures given fewer beads were highly multifunctional and could effectively regress tumors in vivo compared to CAR+Th17 cells activated at a higher bead: T cell ratio. Importantly, we found that medium activation signal strength sustained logarithmic growth of Th17 cells while maintaining a less differentiated phenotype. Furthermore, Th17 cells expanded with fewer beads expressed higher protein concentrations in the Wnt/-catenin and anti-apoptotic signaling pathways via western blot. Additionally, by flow cytometry and XFSeahorse analysis, when Th17 cells were activated with fewer beads they consumed less glucose during activation. This suggests Th17 cells stimulated with fewer beads possess a metabolic profile different than cells activated with more beads. Overall our findings demonstrate that the bead to T cell ratios during activation will dramatically improve next generation ACT clinical trials.
Funding acknowledgments (ACS-IRG, NCI-1 R01 CA 175061, NCI-5 P01 CA154778, NCI-1 R01 CA208514)