RESUMO
Mucosal vaccines have the potential to elicit protective immune responses at the point of entry of respiratory pathogens, thus preventing even the initial seed infection. Unlike licensed injectable vaccines, mucosal vaccines comprising protein subunits are only in development. One of the primary challenges associated with mucosal vaccines has been identifying and characterizing safe yet effective mucosal adjuvants that can effectively prime multi-factorial mucosal immunity. In this study, we tested NanoSTING, a liposomal formulation of the endogenous activator of the stimulator of interferon genes (STING) pathway, cyclic guanosine adenosine monophosphate (cGAMP), as a mucosal adjuvant. We formulated a vaccine based on the H1 antigen (fusion protein of Ag85b and ESAT-6) adjuvanted with NanoSTING. Intranasal immunization of NanoSTING-H1 elicited a strong T-cell response in the lung of vaccinated animals characterized by (a) CXCR3+ KLRG1- lung resident T cells that are known to be essential for controlling bacterial infection, (b) IFNγ-secreting CD4+ T cells which is necessary for intracellular bactericidal activity, and (c) IL17-secreting CD4+ T cells that can confer protective immunity against multiple clinically relevant strains of Mtb. Upon challenge with aerosolized Mycobacterium tuberculosis Erdman strain, intranasal NanoSTING-H1 provides protection comparable to subcutaneous administration of the live attenuated Mycobacterium bovis vaccine strain Bacille-Calmette-Guérin (BCG). Our results indicate that NanoSTING adjuvanted protein vaccines can elicit a multi-factorial immune response that protects from infection by M. tuberculosis.
RESUMO
BACKGROUND: Adoptive cell therapy based on the infusion of chimeric antigen receptor (CAR) T cells has shown remarkable efficacy for the treatment of hematologic malignancies. The primary mechanism of action of these infused T cells is the direct killing of tumor cells expressing the cognate antigen. However, understanding why only some T cells are capable of killing, and identifying mechanisms that can improve killing has remained elusive. METHODS: To identify molecular and cellular mechanisms that can improve T-cell killing, we utilized integrated high-throughput single-cell functional profiling by microscopy, followed by robotic retrieval and transcriptional profiling. RESULTS: With the aid of mathematical modeling we demonstrate that non-killer CAR T cells comprise a heterogeneous population that arise from failure in each of the discrete steps leading to the killing. Differential transcriptional single-cell profiling of killers and non-killers identified CD137 as an inducible costimulatory molecule upregulated on killer T cells. Our single-cell profiling results directly demonstrate that inducible CD137 is feature of killer (and serial killer) T cells and this marks a different subset compared with the CD107apos (degranulating) subset of CAR T cells. Ligation of the induced CD137 with CD137 ligand (CD137L) leads to younger CD19 CAR T cells with sustained killing and lower exhaustion. We genetically modified CAR T cells to co-express CD137L, in trans, and this lead to a profound improvement in anti-tumor efficacy in leukemia and refractory ovarian cancer models in mice. CONCLUSIONS: Broadly, our results illustrate that while non-killer T cells are reflective of population heterogeneity, integrated single-cell profiling can enable identification of mechanisms that can enhance the function/proliferation of killer T cells leading to direct anti-tumor benefit.