Bioluminescence imaging allows measuring CD8 T cell function in the liver

Abstract

In vivo evaluation of CD8 T cell effector (cytotoxic T lymphocyte [CTL]) function in peripheral organs such as the liver is currently not possible but would greatly improve our understanding of local immune regulation, because simple determination of antigen-specific CTL numbers does not predict the outcome of immune responses. In particular, measurement of alanine aminotransferase serum levels is not sensitive enough to detect T cell immunity against low numbers of target hepatocytes. We developed a procedure that detects virus-specific effector function of CTLs in the liver after simultaneous adenoviral transfer of reporter and immune target genes into hepatocytes, followed by bioluminescence imaging of reporter genes. Bioluminescence imaging enabled detection of as few as 10,000 infected hepatocytes in vivo, and even more importantly, quantification of antiviral effector function of as few as 50,000 CTLs. Conclusion: Our results provide evidence that low numbers of antigen-specific CTLs are sufficient to control viral gene expression and eliminate viral infection from hepatocytes. The experimental system established here is a highly sensitive method to simultaneously detect viral infection of hepatocytes and to quantify antiviral CTL function in the liver in vivo and will help in characterizing principles of hepatic immune regulation.

Cutting Edge: The RIG-I Ligand 3pRNA Potently Improves CTL Cross-Priming and Facilitates Antiviral Vaccination

“…Even though OVA-specific Abs were elevated in mice immunized with 3pRNA and OVA (Supplemental Fig. 2G), a role of Abs is unlikely in our model, because OVA is not packaged into the virus during assembly (15), so that OVA-specific Abs cannot neutralize the adenovirus we used. Lysis of infected hepatocytes by OVAspecific Abs would require their binding to the hepatocyte surface, which is unlikely because OVA is a secreted protein.…”

“…Infection was monitored by i.p. injection of Luciferin (50 mmol) and measuring luminescence in anesthetized mice with an IVIS 200 as described (15). Data were analyzed with Living Image 2.50.1 software (Caliper LifeSciences).…”

“…We used a replication-deficient adenovirus expressing luciferase and OVA in infected hepatocytes (15). We immunized wt mice with 3pRNA+OVA or 3pRNA alone.…”

The immune tolerant phase of chronic HBV infection: new perspectives on an old concept

“…Overt T-cell immunity against hepatocytes performed in adenovirus infected mice 31 takes place without alteration in the serum ALT. Furthermore, adoptive transfer of HBV-specific T cells can cause substantial inhibition of HBV replication without increased levels of ALT, an observation that led to the understanding that efficient HBV clearance from infected hepatocytes is also mediated by a cytokine-mediated noncythopathic effect.…”

Immune Response in Hepatitis B Virus Infection

“…Antiviral specific T-cell responses within the liver can be present without any elevation of alanine amino transferase (ALT) levels. Furthermore, quantity and function of HBV-specific T cells correlate with viral control and not with the extent of liver damage (Maini et al 2000b;Stabenow et al 2010). As such, we think that the definition of the "immunotolerant" state in HBV infections requires a better immunological definition and should cover only those subjects who are unable to mount an HBV-specific T-cell response (Bertoletti and Kennedy 2014).…”