UCSF

Over the past few decades, regulatory T cell (Treg) therapy has gained traction for the treatment of autoimmunity, transplant rejection, and other inflammatory diseases. A necessary part of this process is cellular manufacturing, where Tregs are activated with immunomodulatory agents, expanded, and quality assessed for CD4+ and Foxp3+ lineage purity before infusion. A challenge that has emerged in certain patient populations is the failure of Tregs to expand. This clinical manufacturing failure has been associated with the pre-expansion phenotype of the Treg, with a higher proportion of terminally differentiated effector Tregs correlating with poorer expansion. While the heterogeneity of Tregs phenotypic subsets and their expansive capacities has been reported, it is unclear what the required signaling strengths are to induce proliferation within each subset. To investigate this question, we used a biomaterial strategy for precisely attaching Treg stimulatory molecules onto polymeric particles using DNA to act as artificial antigen presenters. First, we optimized the fabrication technique, adapting numerous technologies commonly available to immunology labs, and exemplified its use in both human CD4+ and CD8+ T cell activation. Second, we then applied these materials towards activating human Tregs, identifying unique activation thresholds for inducing expansion and further subset-specific expansion behaviors. Third, unable to drive the expansion of effector Tregs using this approach, we turned to paired single cell RNA and TCR sequencing (scRNA/TCRseq) to identify the distinguishing genes associated with high expansion. Using TCR tracking of highly expanding clones, we were able to subset non-activated cells by their TCR clonotype expansion, enabling the comparison between transcriptomes of high expanders and low expanders. This led to the finding that highly expanding clones were associated with numerous markers associated with Treg stemness, proliferative capacity, and degradation of inhibitory intracellular signals, whereas poorly expanding clones express numerous differentiation and activation genes. Further, we identified a group of proliferative Tregs which displayed an inflammatory phenotype, which prompts further investigation into maintaining Treg purity in strategies that improve expansion. This work sets a foundation in studying Treg subset activation biology using precisely controlled signals and has uncovered numerous gene targets for improving Treg expansion. We believe that investigating these targets and optimizing their activation signals may provide a means of rescuing Treg proliferation in cases of manufacturing failures, reenabling Treg therapy as a possible treatment option for affected patient populations.