Optimal immune activation of naïve CD8 T cells requires signal 1 mediated by the T cell receptor, signal 2 mediated by co-stimulation and signal 3 provided by pro-inflammatory cytokines. However, the potential for signal 3 cytokines to rescue anti-viral responses in functionally exhausted T cells has not been defined. We investigated the effect of using third signal cytokines IL-12 or IFN-α to rescue the exhausted CD8 T cell response characteristic of patients persistently infected with hepatitis B virus (HBV). We found that IL-12, but not IFN-α, potently augmented the capacity of HBV-specific CD8 T cells to produce effector cytokines upon stimulation by cognate antigen. Functional recovery mediated by IL-12 was accompanied by down-modulation of the hallmark inhibitory receptor PD-1 and an increase in the transcription factor T-bet. PD-1 down-regulation was observed in HBV but not CMV-specific T cells, in line with our finding that the highly functional CMV response was not further enhanced by IL-12.IL-12 enhanced a number of characteristics of HBV-specific T cells important for viral control: cytotoxicity, polyfunctionality and multispecificity. Furthermore, IL-12 significantly decreased the pro-apoptotic molecule Bim, which is capable of mediating premature attrition of HBV-specific CD8 T cells. Combining IL-12 with blockade of the PD-1 pathway further increased CD8 functionality in the majority of patients. These data provide new insights into the distinct signalling requirements of exhausted T cells and the potential to recover responses optimised to control persistent viral infections.

Acute liver failure has a high mortality unless patients receive a liver transplant; however, there are insufficient donor organs to meet the clinical need. The liver may rapidly recover from acute injury by hepatic cell regeneration given time. A bioartificial liver machine can provide temporary liver support to enable such regeneration to occur. We developed a bioartificial liver machine using human-derived liver cells encapsulated in alginate, cultured in a fluidized bed bioreactor to a level of function suitable for clinical use (performance competence). HepG2 cells were encapsulated in alginate using a JetCutter to produce ∼500 μm spherical beads containing cells at ∼1.75 million cells/mL beads. Within the beads, encapsulated cells proliferated to form compact cell spheroids (AELS) with good cell-to-cell contact and cell function, that were analyzed functionally and by gene expression at mRNA and protein levels. We established a methodology to enable a ∼34-fold increase in cell density within the AELS over 11–13 days, maintaining cell viability. Optimized nutrient and oxygen provision were numerically modeled and tested experimentally, achieving a cell density at harvest of >45 million cells/mL beads; >5×1010 cells were produced in 1100 mL of beads. This process is scalable to human size ([0.7–1]×1011). A short-term storage protocol at ambient temperature was established, enabling transport from laboratory to bedside over 48 h, appropriate for clinical translation of a manufactured bioartificial liver machine.

No abstract