Microfluidic Squeezing Enables MHC Class I Antigen Presentation by Diverse Immune Cells to Elicit CD8⁺ T Cell...

CD8⁺ T cell responses are the foundation of the recent clinical success of immunotherapy in oncologic indications. Although checkpoint inhibitors have enhanced the activity of existing CD8⁺ T cell responses, therapeutic approaches to generate Ag-specific CD8⁺ T cell responses have had limited success. Here, we demonstrate that cytosolic delivery of Ag through microfluidic squeezing enables MHC class I presentation to CD8⁺ T cells by diverse cell types. In murine dendritic cells (DCs), squeezed DCs were ∼1000-fold more potent at eliciting CD8⁺ T cell responses than DCs cross-presenting the same amount of protein Ag. The approach also enabled engineering of less conventional APCs, such as T cells, for effective priming of CD8⁺ T cells in vitro and in vivo. Mixtures of immune cells, such as murine splenocytes, also elicited CD8⁺ T cell responses in vivo when squeezed with Ag. We demonstrate that squeezing enables effective MHC class I presentation by human DCs, T cells, B cells, and PBMCs and that, in clinical scale formats, the system can squeeze up to 2 billion cells per minute. Using the human papillomavirus 16 (HPV16) murine model, TC-1, we demonstrate that squeezed B cells, T cells, and unfractionated splenocytes elicit antitumor immunity and correlate with an influx of HPV-specific CD8⁺ T cells such that >80% of CD8s in the tumor were HPV specific. Together, these findings demonstrate the potential of cytosolic Ag delivery to drive robust CD8⁺ T cell responses and illustrate the potential for an autologous cell-based vaccine with minimal turnaround time for patients.