RESUMEN
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 â¼1000-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.
Asunto(s)
Presentación de Antígeno , Células Presentadoras de Antígenos/inmunología , Linfocitos T CD8-positivos/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Microfluídica , Neoplasias/inmunología , Traslado Adoptivo , Animales , Células Presentadoras de Antígenos/metabolismo , Antígenos de Neoplasias/inmunología , Linfocitos T CD8-positivos/metabolismo , Técnicas de Cultivo de Célula , Femenino , Humanos , Inmunización , Inmunofenotipificación , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Ratones , Ratones Noqueados , Microfluídica/métodos , Modelos Biológicos , Neoplasias/metabolismo , Neoplasias/patología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismoRESUMEN
Polymorphonuclear neutrophils (PMNs) form the first line of defense against invading microorganisms. We have shown previously that ATP release and autocrine purinergic signaling via P2Y2 receptors are essential for PMN activation. Here we show that mitochondria provide the ATP that initiates PMN activation. Stimulation of formyl peptide receptors increases the mitochondrial membrane potential (Δψm) and triggers a rapid burst of ATP release from PMNs. This burst of ATP release can be blocked by inhibitors of mitochondrial ATP production and requires an initial formyl peptide receptor-induced Ca(2+) signal that triggers mitochondrial activation. The burst of ATP release generated by the mitochondria fuels a first phase of purinergic signaling that boosts Ca(2+) signaling, amplifies mitochondrial ATP production, and initiates functional PMN responses. Cells then switch to glycolytic ATP production, which fuels a second round of purinergic signaling that sustains Ca(2+) signaling via P2X receptor-mediated Ca(2+) influx and maintains functional PMN responses such as oxidative burst, degranulation, and phagocytosis.