Hepatitis (Wellcome)

(Spending a few days in Toronto with my kids, so I can go to the Darwin exhibit at the Royal Ontario Museum.1 Depending on when I get back, my next blog post may be a little delayed.)

Even though cytotoxic T lymphocytes are called “cytotoxic”, it was no surprise when new techniques in the mid-90s suggested that CTL might have other strings to their bows. (I talked about this the other day.) Some experiments were also pointing the same way. For example, Frank Chisari’s group offered evidence that CTL might be able to control hepatitis B virus without actually being cytotoxic.

Hepatitis B virus was a particularly difficult case for study in general, because it’s pretty much a human-specific virus. (HBV is a pain in tissue culture. There are a couple of animal models, but they’re hardly convenient. I mean, ducks? Woodchucks?) From studies of the natural disease, it seemed pretty clear that natural HBV infection was often handled by CTL; people who control HBV show a strong CTL response, those in whom HBV progresses and becomes chronic usually do not. But testing the function of CTL in HBV infection wasn’t easy without an animal model.

So Chisari made an animal model. He made transgenic mice that express the HBV genome (that is, the mouse genome contained the HBV genome) under a liver-specific promoter.2 There you go, hepatitis B virus in the mouse liver. It can’t spread and infect new cells as would normally happen in humans, but on the other hand you’re expressing the virus in essentially all the liver cells anyway, so you don’t need to infect new cells.

Hepatitis B virus-transgenic mice

Of course, the “virus” is “self” under those conditions, meaning that the immune system doesn’t react to it. So Chisari’s group immunized other mice to raise HBV-specific CTL, and transferred the CTL into the transgenic mice.3 These HBV-specific CTL did two things: They apparently controlled the virus “infection”, and they damaged the liver.4 The presumption was that the two findings were the same effect, and the CTL were trying to eliminate virus by killing liver cells: “Our data show that antigen-specific immune effector mechanisms can destroy HBV-positive hepatocytes in vitro and in vivo … “.

Hepatitis viruses (Wellcome)
Hepatitis viruses

Six years later, though, they were suggesting the opposite, demonstrating that in fact CTL were controlling the “virus” through a non-cytolytic mechanism.5 (Actually, they showed very similar data a couple of years earlier than that,6 but the 1996 paper went further with the mechanisms and was overall more solid.) Essentially, they demonstrated that CTL were shutting down virus expression in the liver by releasing interferon and other cytokines. There is some liver damage, but it’s not necessarily because of direct cytotoxity; it’s a side-effect of the cytokines, not directly related to the viral clearance. (Recently, it’s been proposed that the CTL make a decision which route to go — cytotoxicity vs. cytokine-mediated control — based on the amount of virus antigen. 7 This might offer a way to manipulate this and drive the response to the most effective system in other infections, though it’s far from trivial to adjust amount of viral antigen.)

Non-cytotoxic control of HBV

So what? What difference does it make how the CTL are clearing virus, so long as they do clear it? Here are some reasons to care:

  • This way a few CTL can affect many target cells. There are some 1011 hepatocytes, and hepatitis viruses can infect a lot of them, very fast. There are different estimates for how long it takes for a CTL to deliver a lethal hit when it’s being cytotoxic, but let’s say something like 30 – 60 minutes per cell. That’s a long, long time for CTL to kill off all the infected cells. If all they have to do is release interferon, they can probably pick off many cells at once and move on. This is a faster way to control viruses, and it doesn’t take as many CTL. Guidotti et al transferred 5 x 106 HBV-specific CTL into the transgenic mice; within 24 hours, all of the virus had been shut down (remember. these are transgenic mice expressing “virus” in every liver cell).
  • Again: There are some 1011 hepatocytes, and hepatitis viruses can infect a lot of them, very fast. If CTL have to kill all the virus-infected cells, what’s left of the liver to do it’s usual liverly duties? If the infection can be shut down without killing the cells, you’ve saved your liver. Those 5 x 106 CTL shut down the virus without killing more than 10% of the liver cells.
  • Potentially, any inflammation in the liver can lead to protection. If you have HBV, and you’re infected by lymphocytic choriomeningitis, (well, first you’d likely be a mouse), the inflammation induced by the LCMV might shut down the HBV, as a handy side effect. This cross-protection from other viruses has actually been seen both in mice8 and — maybe — in humans as well.9

So the finding that CTL are capable of shutting down virus replication without having to kill the infected cells, fit very nicely with the new technology showing that CTL commonly have these mechanisms available.

  1. Also I promise it’s a complete coincidence that my Red Sox were playing the Blue Jays here last few days. The Jays gave them a whuppin’, but William and I had a fine time at the ballpark anyway.[]
  2. Chisari, F. V., Pinkert, C. A., Milich, D. R., Filippi, P., McLachlan, A., Palmiter, R. D., and Brinster, R. L. (1985). A transgenic mouse model of the chronic hepatitis B surface antigen carrier state. Science 230, 1157-1160.[]
  3. As you can see, the system is pretty elaborate, and I’ve never really felt all that comfortable with it — just too Rube Goldberg-ish for my liking — even though there’s nothing specific I can point to; and Chisari’s group is conscientious about their controls.[]
  4. Moriyama, T., Guilhot, S., Klopchin, K., Moss, B., Pinkert, C. A., Palmiter, R. D., Brinster, R. L., Kanagawa, O., and Chisari, F. V. (1990). Immunobiology and pathogenesis of hepatocellular injury in hepatitis B virus transgenic mice. Science 248, 361-364.[]
  5. Guidotti, L. G., Ishikawa, T., Hobbs, M. V., Matxke, B., Schreiber, R., and Chisari, F. V. (1996). Intracellular inactivation of the hepatitis B virus by cytotoxic T lymphocytes. Immunity 4, 25-36.[]
  6. Guidotti, L. G., Ando, K., Hobbs, M. V., Ishikawa, T., Runkel, L., Schreiber, R. D., and Chisari, F. V. (1994). Cytotoxic T lymphocytes inhibit hepatitis B virus gene expression by a noncytolytic mechanism in transgenic mice. Proc Natl Acad Sci U S A 91, 3764-3768.
    Tsui, L. V., Guidotti, L. G., Ishikawa, T., and Chisari, F. V. (1995). Posttranscriptional clearance of hepatitis B virus RNA by cytotoxic T lymphocyte-activated hepatocytes. Proc Natl Acad Sci U S A 92, 12398-12402.[]
  7. Gehring, A. J., Sun, D., Kennedy, P. T., Nolte-‘t Hoen, E., Lim, S. G., Wasser, S., Selden, C., Maini, M. K., Davis, D. M., Nassal, M., and Bertoletti, A. (2007). The level of viral antigen presented by hepatocytes influences CD8 T-cell function. J Virol 81, 2940-2949.[]
  8. Guidotti, L. G., Borrow, P., Hobbs, M. V., Matzke, B., Gresser, I., Oldstone, M. B., and Chisari, F. V. (1996). Viral cross talk: intracellular inactivation of the hepatitis B virus during an unrelated viral infection of the liver. Proc Natl Acad Sci U S A 93, 4589-4594.[]
  9. Thio, C. L., Netski, D. M., Myung, J., Seaberg, E. C., and Thomas, D. L. (2004). Changes in hepatitis B virus DNA levels with acute HIV infection. Clin Infect Dis 38, 1024-1029.[]