Exploring immune modulating strategies to restore antiviral effector functions of intrahepatic CD8 T cells
Final Report Abstract
1.4HBV-Smut transgenic (tg) mice harbour a replicating 1.4 HBV genome in the liver. Tg hepatocytes express endogenous large/middle surface and core antigens and efficiently secrete precore (HBV-E) but not ´empty´ S-particles or infectious HBV virions. We have speculated that the absence of circulating S-particles could facilitate priming of surface antigen-specific CD8+ T-cell responses. However, we could induce antiviral core (Kb/C93)- but not surface (Kb/S190)-specific CD8+ T-cell responses in 1.4HBV-Smut tg mice by DNA-based vaccines. In this project, we identified novel interferences between HBV-surface antigen derived epitopes (operating in antigen expressing hepatocytes and APCs targeted by plasmid DNA injection) that establish ´immunodominance´ hierarchies and modulate the induction of multispecific CD8 T-cell responses: A Kb/S190-mediated ´immunodominance´ operating in surface antigen-expressing cells (endogenous pathway) but not in rSP-pulsed cells (exogenous pathway), led to an efficient suppression of the presentation of a low affine Kb/S208 epitope and to a significant suppression of Kb/S208-specific CD8 T-cells by DNA-based vaccines. This Kb/S190-mediated ´immunodominance´ also operated in 1.4HBV-Smut tg hepatocytes in vivo and efficiently suppressed the presentation of the Kb/S208 epitope. As a consequence, we could prime non-functional IFN-γ+ Kb/S208-specific CD8+ T cells in 1.4HBV-Smut tg mice by DNA- and protein-based vaccines. This suggested that epitope presentation on virusharboring hepatocytes has a strong impact on the priming of antiviral CD8+ T-cell responses. We further characterized the antigen requirements for priming antiviral HBV-C-specific effector CD8+ T cells in 1.4HBV-Smut tg mice. The HBV-C protein contains a COOH-terminal cationic domain (C150-183) that is crucial for binding immune-stimulatory heterologous RNA in antigen-producing bacteria. To investigate whether this domain has an impact on the RNA-binding and immunogenicity of endogenously expressed HBV-C, we evaluated induction of Kb/C93-specific CD8+ T-cell responses in B6 and 1.4HBV-Smut tg mice by mutant core antigens and developed a novel expression/purification system for HBV-C/RNA particles in vector-transfected HEK-293 cells. We showed that HBV-C but not HBV-C149 particles (lacking the cationic domain) encapsidate mammalian RNA. Prevention of specific serine-phosphorylation at three repeated SPRRR motifs, either by exchanging the entire cationic domain with an unrelated HIV-tat48-57-like sequence (HBV-C149tat) or by exchanging the serine residues S155, S162 and S170 with alanines (HBV-CAAA), significantly enhanced the RNA- binding of particles. An assembly-deficient HBV-Cmut∆113-119 antigen, associated with stress-proteins and the mature form of complement component 1Q subcomponent-binding protein, also bound cellular RNA. All vector constructs efficiently induced Kb/C93-specific CD8+ T-cells in B6 mice. However, only the particle-forming and RNA-binding HBV-C, HBV-CAAA and HBV-C149tat antigens elicited Kb/C93-specific CD8+ T-cells in 1.4HBV- Smut tg mice and inhibited HBV replication in the liver. The encapsidation of cellular RNA into particles, but not binding to monomeric core subunits, was thus crucial to elicit an antiviral CD8+ T-cell immunity in 1.4HBV-Smut tg mice. Furthermore, particle-bound mammalian RNA functioned as TLR-7 ligand and stimulated a Th1-biased humoral immune response in B6 but not in TLR-7-/- mice. The HBV-C-specific trapping of an endogenous RNA- based ´adjuvant´ activity could be a promising platform technology to induce antiviral or antitumor CD8+ T cells under stringent conditions (e.g., operating against the tolerogenic milieu of an antigen expressing liver) by chimeric DNA-based vaccines.
Publications
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(2011) Strict control of transgene expression in a mouse model for sensitive biological applications based on RMCE compatible ES cells. Nucleic Acids Res. Jan;39(1):e1
Sandhu U, Cebula M, Behme S, Riemer P, Wodarczyk C, Metzger D, Reimann J, Schirmbeck R, Hauser H, Wirth D
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(2013) An inducible transgenic mouse model for immune mediated hepatitis showing clearance of antigen expressing hepatocytes by CD8+ T cells. PLoS One. Jul 15;8(7):e68720
Cebula M, Ochel A, Hillebrand U, Pils MC, Schirmbeck R, Hauser H, Wirth D
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(2014) Differential presentation of endogenous and exogenous hepatitis B surface antigens influences priming of CD8(+) T cells in an epitope-specific manner. Eur J Immunol. Jul;44(7):1981-91
Riedl P, Reiser M, Stifter K, Krieger J, Schirmbeck R
