Abstract: This proposal aims to develop and optimize antigen-specific T cell activation and expansion platforms for antigen-specific immunotherapy. Our goal is to enable the generation of new cancer immunotherapies and reduce time, complexity, and cost while improving product performance and consistency. Our focus is on harnessing both CD4+ and CD8+ immune responses to enhance cancer immunotherapy, although the technologies developed are expected to have broader applications in vaccine development and autoimmunity as well. In SA1 we will engineer nanoparticle-based artificial antigen-presenting cells (aAPCs) to activate and expand CD4+ T cells. We aim to manipulate two key CD4+ T cell phenotypes: cytotoxic CD4+ T cells and CD4+ T cells that help effector CD8+ T cells. Various features of the aAPCs will be optimized, such as mechanical stiffness, cytokines, T cell receptor affinity, and signal 2 effects. This optimization process will include analyzing cytotoxic granule production, RNA-seq transcriptional profiles, and antitumor activity in murine tumor models. Additionally, a prototype class I/II aAPC that activates both CD4+ and CD8+ cells will be developed. In SA2 we will engineer biodegradable hydrogel microparticle (MP)-based aAPCs for T cell stimulation that has both ex vivo and in vivo applications. We aim to create aAPCs that incorporate T cell-stimulating signals, biomechanical cues, and key biological features of the lymph node. The phenotype and function of the engineered T cells will be examined through adoptive transfer. Furthermore, the ability of the MP aAPCs to activate T cells in vivo and their functional efficacy in murine tumor models will be analyzed. SA3 involves applying the developed aAPC platforms to human T cells. Human leukocyte antigen class II (HLA II)-based nanoparticle and microparticle aAPCs will be used to induce cytotoxic CD4+ T cells ex vivo, and their functional activity against tumor cell lines will be evaluated in xenogeneic humanized mouse models. Combined HLA I/II aAPCs will be used to co-activate human CD8+ and CD4+ T cells, and their lytic activity, antitumor function. Overall, this proposal aims to advance the field of antigen-specific immunotherapy by developing innovative T cell activation platforms that have the potential to improve therapeutic outcomes in cancer and other diseases. The deliverables of this project include nanoparticle aAPCs targeting CD4+ cytotoxic T cells, combinatorial MHC I/II aAPCs for simultaneous activation of CD4+ and CD8+ cells, hydrogel microparticle-based aAPCs capable of activating and expanding CD8+ and CD4+ T cells, and human HLA II-based nanoparticle and microparticle aAPCs, as well as combined HLA I/II aAPCs for co-activation of human CD4+ and CD8+ T cells.