TLR9-Mediated Conditioning of Liver Environment Is Essential for Successful Intrahepatic Immunotherapy and Effective Memory Recall

Abstract

Immune defense against hepatotropic viruses such as hepatitis B (HBV) and hepatitis C (HCV) poses a major challenge for therapeutic approaches. Intrahepatic cytotoxic CD8 T cells that are crucial for an immune response against these viruses often become exhausted resulting in chronic infection. We elucidated the T cell response upon therapeutic vaccination in inducible transgenic mouse models in which variable percentages of antigen-expressing hepatocytes can be adjusted, providing mosaic antigen distribution and reflecting the varying viral antigen loads observed in patients. Vaccination-induced endogenous CD8 T cells could eliminate low antigen loads in liver but were functionally impaired if confronted with elevated antigen loads. Strikingly, only by conditioning the liver environment with TLR9 ligand prior and early after peripheral vaccination, successful immunization against high intrahepatic antigen density with its elimination was achieved. Moreover, TLR9 immunomodulation was also indispensable for functional memory recall after high frequency antigen challenge. Together, the results indicate that TLR9-mediated conditioning of liver environment during therapeutic vaccination or antigen reoccurrence is crucial for an efficacious intrahepatic T cell response.

Outcome of Antiviral Immunity in the Liver Is Shaped by the Level of Antigen Expressed in Infected Hepatocytes

“…Therefore, we combined the known hepatotropism of adenoviruses with hepatocyte‐selective gene expression through the TTR promoter, similar to the combination of hepatotropism and hepatrophism used by HBV . The outcome of infection by these otherwise identical viruses was completely different: Ad‐CMV‐GOL infection was efficiently cleared, whereas infection with Ad‐TTR‐GOL persisted, independent of the numbers of infected hepatocytes covering a 4log 10 range of infection dose from 10 5 to 10 9 PFU/mouse, thus going far beyond the range of antigen‐expressing hepatocytes studied so far . Linking ovalbumin, EGFP, and luciferase genes by 2A sequences in recombinant adenoviruses results in equimolar gene expression and allows to quantitatively and most sensitively detect infection and immune‐mediated clearance of infected hepatocytes by in vivo bioluminescence imaging .…”

“…This was only related to the different promoters driving the different expression levels of genes delivered through adenoviral vectors. Previously, high numbers of antigen‐expressing hepatocytes were claimed to cause CD8 T‐cell dysfunction, although only a range between 10% and 50% of antigen‐expressing hepatocytes were investigated . In a humanized murine model, the load of HBV antigens was determined to influence antiviral immunity .…”

“…Previously, high numbers of antigen-expressing hepatocytes were claimed to cause CD8 T-cell dysfunction, although only a range between 10% and 50% of antigen-expressing hepatocytes were investigated. (24,26,27) In a humanized murine model, the load of HBV antigens was determined to influence antiviral immunity. (28) However, in these mice the human liver graft and the human immune system were not human leukocyte antigen (HLA)matched, rendering it unlikely to detect HLA-specific T-cell responses.…”

Advances in Targeting the Innate and Adaptive Immune Systems to Cure Chronic Hepatitis B Virus Infection

“…Therefore, the antiviral effects of HBV-specific T cells may require appropriate conditions in the liver. TLR agonists may be useful for the promotion of T cell functions in the liver (160,164,165), by recruiting various immune cells into the liver to form tertiary lymphoid structures (166)(167)(168). These aspects have been discussed in recent reviews and need to be investigated in future studies (32,52,58,92).…”

Outcome of anti-viral immunity in the liver is shaped by the level of antigen expressed in infected hepatocytes

“…(23) The outcome of infection by these otherwise identical viruses was completely different: Ad-CMV-GOL infection was efficiently cleared, whereas infection with Ad-TTR-GOL persisted, independent of the numbers of infected hepatocytes covering a 4log 10 range of infection dose from 10 5 to 10 9 PFU/mouse, thus going far beyond the range of antigen-expressing hepatocytes studied so far. (24) Linking ovalbumin, EGFP, and luciferase genes by 2A sequences in recombinant adenoviruses results in equimolar gene expression and allows to quantitatively and most sensitively detect infection and immune-mediated clearance of infected hepatocytes by in vivo bioluminescence imaging. (22,25) Use of replication-deficient adenoviruses allows us to focus on the antigens expressed, as no adenoviral antigens/epitopes are expressed/presented.…”