Mystery Rays from Outer Space

Hepatitis

(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

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.
   also
   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.[↩]

Breart et al, Fig. 3. Direct action of CTLs on individual tumor cells drives tumor regression

Intravital microscopy — microscopic analysis, in real time, of processes within a living animal — has been used in immunology for maybe a decade now, but it hasn’t lost its cool factor yet. I don’t know that there have been any great intellectual breakthroughs arising from the work, but we have learned a fair bit about, say, interactions between T cells and their targets, and migration patterns, and so on. And of course there’s a huge help in visualization, which undoubtedly helps people understand what’s happening and, hopefully, develop other experimental approaches to test it.

Just as importantly, the “Awesome” factor of these things is absolutely off the scale. I pointed out Uli von Andrian’s collection of intravital videos the other day, and now the latest issue of Journal of Clinical Investigation has a paper from Phillipe Bousso’s group, showing 2-photon microscopy of cytotoxic T lymphocytes attacking a tumor.

The paper is:

Breart, B., Lemaître, F., Celli, S., Bousso, P. (2008). Two-photon imaging of intratumoral CD8+ T cell cytotoxic activity during adoptive T cell therapy in mice. Journal of Clinical Investigation, 118(4), 1390-1397. DOI: 10.1172/JCI34388

They used the EL4/EG7 tumor model in mice. These cells form solid tumors in C57BL/6 mice, and are not rejected by the immune system. The EG7 cells are derived from EL4; they have had a defined antigen introduced, and if you transfer activated T cells against the antigen, the tumor will be rejected. If you transfer naive T cells, and depend on them to be activated by the tumor, you’re out of luck; the tumor is not rejected. ¹ They were able to watch all these things happening, in real time.

Here’s what happens with activated CTL (orange) around a tumor site (tumor cells in yellow/greenish). Watch the CTL zipping around merrily in areas where there are no tumor cells, and then screeching to a halt as they identify tumor antigen, engage their targets, and begin to kill:

(Embedded video! I’m so MySpace! I’m going to use tripple exclamation marks and mispell lot’s of words!!!)

The article is free access, I believe, so you should check it out for yourself; there are five videos to watch in the supplemental data. They show CTL engaging tumor cells and actively killing them, using indicators for cell death so they don’t have to guess what’s happening.

I think this is mainly a technical tour de force, and the amount of new information about tumor immunology is relatively small. But there are a couple things of interest. One is that naive T cells — the guys who do not reject the tumor — seem kind of indifferent to the whole thing. It’s not a question of the CTL entering the tumor, and then being turned off (which would have been my guess); rather, the naive cells never even entered the tumor in the first place:

Although CTL infiltration was quite variable in the different regions of the tumor (Figure 6A), EG7 patches were eliminated in CTL-rich areas, which was evidence that in vivo primed CTLs were not grossly impaired in their ability to kill target cells … Thus, the low level of CD8⁺ T cell infiltration, rather than a defect in the cytotoxic activity, appeared to be responsible for the inefficient response mounted by in vivo primed OT-I T cells.

Another surprising finding — which is so different from previous work in different systems that I’m hesitant to believe it — is the timing of cell killing. Previous studies (such as the von Andrian paper² that produced this video) have suggested that CTL kill their targets in something under an hour; maybe 30 minutes or even less. Here. Bousso’s group find that the tumor cells take something like 6 hours to be killed. That’s such a large difference — and has such important implications for effectiveness of CTL killing — that, as I say, I’d like to see it confirmed before I take it to the bank.

Bousso’s web site has a bunch of other equally fascinating videos; check them all out.

1. This is probably related to the ability of tumors to suppress immune responses, which I’ve talked about before.[↩]
2. Mempel, T. R., Pittet, M. J., Khazaie, K., Weninger, W., Weissleder, R., von Boehmer, H., and von Andrian, U. H. (2006). Regulatory T cells reversibly suppress cytotoxic T cell function independent of effector differentiation. Immunity 25, 129-141.[↩]

In one of those bizarre twists of logic, cytotoxic T lymphocytes were so named because they’re T lymphocytes that are cytotoxic. Is that all they are?

Cytotoxicity is relatively easy to measure — there are straightforward ways to measure cell death, and it can be a nice, binary, black/white distinction. If you take lymphocytes from a mouse (or a person) that was previously infected with a virus, and you mix those lymphocytes with cells infected with the same virus, the infected cells will be killed. If you look at the surface markers of the cells responsible for the killing, you can narrow it down to T cells (i.e. with the T cell receptor) that have the CD8 surface marker. ¹

⁵¹Cr release assays² are a traditional way of measuring cell death, and you can set them up in 96-well plates and get moderate throughput to test multiple conditions. It’s a convenient system, and it was the first one to be widely used to T cells. Doherty and Zinkernagel used it in their Nobel-Prize winning work on MHC restriction, for example.

However, as you’d expect, systems which are designed for operator convenience don’t necessarily reflect reality. Measuring cell death in vivo, that is, inside a virus-infected animal, is much more complicated than in a 96-well plate. Do CTL actually kill in that context? And even if it does happen, is it the only thing that happens? Could CTL be doing something else during an infection, other than killing, that helps in their mission?

You might wonder if immunologists were blinkered by the name — how could cytotoxic T lymphocytes not be, first and foremost, cytotoxic? — but I don’t think it’s revisionist to say that’s not true. I think most of us were pretty sure that CTL had lots of other weapons in their arsenal, but how much other stuff? How often were CTL actually cytotoxic, and how often did they do other stuff?

One problem with cytotoxicity as an assay for this question, is that it’s a bulk assay. Until recently you couldn’t really measure killing by a single CTL. (You can now, though. Uli von Andrian has some beautiful videos of CTL punching holes in their targets here here, from his 1996 2006 Immunity paper. ³ Von Andrian’s site is filled with beautiful and amazing videos; check them out.). You mix together a batch of CTL with the targets — the targets die, well and good — but were all of the specific CTL helping out, or was it just the work of a minority of them that are specialized for killing?

In 1996, Mark Davis introduced a new and exciting technology, MHC tetramers, that’s able to rapidly identify T cells by phenotype rather than function. ⁴ That is, if a T cell has the right T cell receptor to recognize a virus-infected cell, tetramers can show you the T cell — even if it cannot kill. This was pretty revolutionary, and led to some drastic increases in estimates of T cell number — previous methods of counting specific T cells were known to be underestimates, and tetramer staining showed us that there were sometimes 100 or 1000 times more CTL floating around than had been esimated.

It didn’t really answer the cytotoxicity question, though. Tetramer staining correlates well with cytotoxicity levels, but you’d see that even if 1% of the CTL were actually cytotoxic, and the rest were doing something else.

Another new technique that came out around the same time or a little later⁵ is intracellular cytokine staining. This identifies T cells that not only recognize their target, but react to it by producing cytokines, such as interferon. In other words, intracellular cytokine staining not only lets you measure T cells, it offers a measure of functionality other than cytotoxicity. Correlating this with tetramer staining was a little more informative; most tetramer-positive cells were also able to produce interferon, for example.

So we know that there are a lot of CTL; we knew that most produced interferon and other cytokines when stimulated. But — to finally get to the point — we also knew even by the that cytotoxicity is important. Just about the same time as all these other assays were coming out, a perforin knockout mouse was made. Perforin is a protein that’s believed to be important in CTL cytotoxicity and not much else.⁶ Even though other proteins are also involved in cytotoxicity, mice without perforin weren’t able to clear lymphocytic choriomeningitis virus the way wild-type mice did. ⁷

So what’s the interferon there for? Interferon isn’t directly involved in cytotoxicity, and experiments from around that time showed that CTL can do a lot of antiviral work just using interferon, without getting all cytotoxic on us. I was originally going to talk about that experiment — Frank Chisari’s hepatitis B mouse model — here, but this is all background, so I’ll get to that some other day.

1. Also, probably, you’ll find that natural killer, NK, cells do some killing too.[↩]
2. Which really suck, but they’re better than the alternatives, which suck even more[↩]
3. Mempel, T. R., Pittet, M. J., Khazaie, K., Weninger, W., Weissleder, R., von Boehmer, H., and von Andrian, U. H. (2006). Regulatory T cells reversibly suppress cytotoxic T cell function independent of effector differentiation. Immunity 25, 129-141. doi:10.1016/j.immuni.2006.04.015 [↩]
4. Altman, J.D., Moss, P.A., Goulder, P.J., Barouch, D.H., McHeyzer-Williams, M.G., Bell, J.I., McMichael, A.J., Davis, M.M. (1996). Phenotypic Analysis of Antigen-Specific T Lymphocytes. Science, 274(5284), 94-96. DOI: 10.1126/science.274.5284.94[↩]
5. I confess I’m not quite sure when it was developed — it became popular in the mid- to late-90s, is all I remember. The assay is basically a spinoff of the earlier ELISPOT assay that’s been adapted to flow cytometry, and ELISPOT assays were being used in the early 1990s.[↩]
6. I know it’s debatable, but that’s close enough for a first approximation[↩]
7. Walsh, C. M., Matloubian, M., Liu, C. C., Ueda, R., Kurahara, C. G., Christensen, J. L., Huang, M. T., Young, J. D., Ahmed, R., and Clark, W. R. (1994). Immune function in mice lacking the perforin gene. Proc Natl Acad Sci U S A 91, 10854-10858.andKagi, D., Ledermann, B., Burki, K., Seiler, P., Odermatt, B., Olsen, K. J., Podack, E. R., Zinkernagel, R. M., and Hengartner, H. (1994). Cytotoxicity mediated by T cells and natural killer cells is greatly impaired in perforin-deficient mice. Nature 369, 31-37.[↩]

Sand rat, Psammomys obesus

Although a lot of viruses have ways of blocking recognition by T cells and NK cells, there’s not much known about the importance of these mechanisms in actual infections. That’s because the best-studied viruses in this class tend to be highly species-specific. So, for example, we don’t have good animal models for the human herpesviruses human cytomegalovirus, Epstein-Barr virus, varicella-zoster virus, or Kaposi’s sarcoma herpesvirus. Herpes simplex virus does infect mice, but its immune evasion molecule ICP47 doesn’t work well in mice, so we’re no further ahead.¹

Immune evasion by all these viruses has been studied pretty extensively in cultured cells, but because they essentially only infect humans we only have circumstantial evidence for a role in vivo. Similarly, porcine, bovine, and equine herpesviruses encode immune evasion molecules, but pigs, cattle, and horses are not very convenient models for basic research either.Another major group of viruses, besides the herpesviruses, that are noted for immune evasion are the adenoviruses. However, only the human (and primate) adenoviruses contain the classical E3gp19k immune evasion molecule. There’s an animal model for human adenoviruses (the cotton rat) but as I pointed out the other day, there’s little evidence for an important function of CTL immune evasion in this model.

So what we need are small animal, and preferably lab mouse, models for infection with adenoviruses or herpesviruses that include immune evasion molecules. As far as we know, mouse adenoviruses don’t have T cell or NK cell immune evasion properties. That leaves us with mouse herpesviruses. Of the hundreds of known herpesviruses, six are known to be murid-specific (see the table at right), and three of those infect lab mice. One of those is totally obscure (there’s very little known about mouse thymic herpesvirus), leaving us with mouse cytomegalovirus and mouse herpesvirus 68 (MHV68). I’ve already commented on immune evasion by mouse CMV. The bottom line is that removing all known T cell evasion molecules from MCMV makes almost no difference to infection or latency; the one difference is that the virus persists longer and at higher levels in salivary glands. That may be important in virus transmission, but lacks a little oomph.

That leaves MHV68, and I’m pleased to say that there is actually some evidence that T cell immune evasion is important for this guy. (I’ve mentioned this in passing earlier, but it deserves its own post.) MHV68 uses a gene “mK3″ to attack MHC class I (MHC class I is recognized by cytotoxic T lymphocytes). ² In 2002, Philip Stevenson and Stacey Efstathiou made a mutant of MHV68 lacking mK3,³ and tested its ability to infect mice:

Stevenson, P., May, J., Smith, X., Marques, S., Adler, H., Koszinowski, U., Simas, J., Efstathiou, S. (2002). K3-mediated evasion of CD8+ T cells aids amplification of a latent γ-herpesvirus. Nature Immunology DOI: 10.1038/ni818

In cultured cells, where there’s no immune system, the mutant virus grows exactly as well as wild-type MHV68. As well (more surprisingly) there was no difference in the initial virus clearance; the mutant virus and the parent were both cleared from the lungs of infected mice at the same rate, and were undetectable after about 13 days. However (finally!) there was a big difference in the amount of latency. The figure to the right shows latent wild-type (left panel) and mutant (right panel) MHV68, the black dots, in spleens of mice infected 13 days previously. What’s more, during the latent phase there was a better immune response to mutant MHV68; mice infected with the mutant virus had about twice as many CTL specific for MHV68.

The association of higher virus-specific CTL frequencies with lower viral loads suggested that CTLs were responsible for the elimination of DeltaK3 viruses during latency amplification.

Eliminating CTL from the mice removed the difference; in the absense of CTL, the mK3 knockout virus established latency just as well as the wild-type. This shows that effects on CTL, and probably not some other unknown function of mK3, are responsible for the difference.

So with the two and a half authentic models of infection (I count the herpes simplex virus one as a half because it’s more contrived than a natural infection) we have immune evasion molecules helping to establish latency (MHV68), helping to reactivate from latency (herpes simplex) and helping with persistence (MCMV) . In no case is there much effect on acute infection.

1. However, swapping in a different immune evasion molecule, that does work in mice, helps HSV reactivate from latency; see my previous post here. [↩]
2. mK3 is so named because it’s highly similar to a Kaposi’s sarcoma herpesvirus gene called “K3″; K3 is one of two KSHV genes that target MHC class I, but we don’t know much about KSHV infection. MHV68 probably isn’t a great model for KSHV, in spite of using a similar protein in immune evasion.[↩]
3. This virus was still able to down-regulate MHC class I to some extent, though, so there may be still other immune evasion genes in MHV68[↩]

I’ve lost an old friend. Apparently it’s been dead for quite a while, but I just found out about it:¹ E3gp19k doesn’t protect against pulmonary inflammation in cotton rats. A moment of silence, please.

The “friend” in question is the paper
Ginsberg, H., S, Lundholm-Beauchamp, U., Horswood, R., L, Pernis, B., Wold, W., S, Chanock, R., M, and Prince, G., A (1989). Role of early region 3 (E3) in pathogenesis of adenovirus disease. Proceedings of the National Academy of Sciences of the United States of America Proc Natl Acad Sci U S A 86, 3823-3827.

I’ve been using it for years to illustrate a particular point. Unfortunately, while grinding through the literature a couple of weeks ago, I discovered that the paper’s main conclusion was undercut in 1994, and then again in 2002. No one else seems to have noticed this either, so at least I have lots of company.

Viruses have immune evasion genes that allow them to escape or resist the immune response; they target all aspects of the immune system, from interferon to antibodies to cytotoxic T lymphocytes (CTL) to natural killer (NK) cells. I’m particularly interested in the class of viral immune evasion molecules that target CTL recognition by blocking MHC class I antigen presentation. (A summary of antigen presentation is here.) However, as I’ve pointed out before, there is surprisingly little evidence that these genes are important in actual infections, as opposed to their effect in cultured cells. This contrasts with the clear and striking evidence that cytokine escape is a critical virulence factor for several viruses.

CTL evasion in vivo
One big stumbling block in analyzing the importance of antigen presentation blockade in viral infection is that the viruses that have developed this approach tend to be highly species-specific: they’re herpesviruses and adenoviruses. (And HIV; same deal as far as species specificity.) Because, for obvious reasons, research has focused on human pathogens, it’s been hard to move cell culture results into in vivo studies. You don’t see volunteers lining up to be infected with mutant herpes simplex virus, and even where lab animals can be infected with one of these viruses (e.g. herpes simplex) the immune evasion may not work in the lab animal, or there may be some other difference in the infection that makes interpretation difficult.

There are a couple of herpesvirus models. Mouse cytomegalovirus and mouse herpesvirus 68 are both natural mouse pathogens with authentic CTL immune evasion systems. In these cases there isn’t a whole lot of effect from the MCMV CTL evasion molecules (reduced virus titer in salivary glands), and there’s a moderate effect from MHV68 CTL evasion (reduced establishment of latency).

There are in fact a bunch of animal adenoviruses, but — strangely — none of these seem to have CTL evasion molecules. At any rate, none of the dozen or so whose genomes I’ve looked at share the human E3gp19k protein that’s long been shown to block MHC class I antigen processing. In particular, mouse adenovirus 1 does NOT block antigen presentation, at least as determined in one careful study. And while human adenoviruses will just about infect mice, it’s not a productive infection. The human adenoviruses do express some genes in mice, but they don’t efficiently go all the way through replication. It’s not a good model for the natural infection, and so there was some interest, 25-odd years ago, when an animal model for human adenovirus infection was identified.

This is the cotton rat, Sigmodon hispidus. It turns out that at least some human adenoviruses, including the popular type 5, replicate quite well in cotton rats and establish a pneumonia that is vaguely reminiscent of the human disease.² The system has never really become very popular, probably because cotton rats are vicious, evil little bastards that are as much wolverine as rodent. You handle them with steel-mesh gloves, and it’s still a sporting proposition as to whether the researcher or the rat draws first blood. Subsequent experiments showed that in fact the disease in mice looked quite similar to that in cotton rats, suggesting that mice were an adequate model after all,³ and there was an audible sigh of relief as researchers went back to peaceful little mice again.

Adenovirus immune evasion in cotton rats
But before the boom was over, Harry Ginsberg’s group tested what happened when you infect cotton rats with adenoviruses with, or without, the CTL evasion gene E3gp19k. The Ad5 E3 region is mostly involved in immune evasion of various kinds — cytokines as well as CTL — and as such it’s non-essential in cultured cells, where there’s no immune system. It’s not unusual for spontaneous E3 deletions to pop up in virus stocks passaged in tissue culture. Ginsberg infected cotton rats with some of these mutants. Overall, the mutants spread and replicated in the rats just as well as wild-type virus did. But there was one big difference. Here’s the money shot (Ginsberg et al, Fig. 2; click for a larger version):

These are cotton rat lungs. On the left, infected with wild-type virus; and on the right, infected with a deletion mutant virus lacking E3gp19k. There’s much more infiltrate (inflammation) in the lungs on the right. The obvious explanation, and the one I’ve used for nearly 20 years, is that E3gp19k actually protects the host as much as the virus. In many cases (especially in pneumonia), it’s inflammation that causes the clinical signs of disease. By reducing CTL recognition of infected cells, E3gp19k reduces inflammation and should reduce the amount of disease.

There are some puzzling parts of this story (why E3gp19k, instead of, say, 14.7k, which reduces cytokines and should have a larger effect on inflammation?) and some weaknesses in the paper (they never actually showed that E3gp19k works in cotton rat cells, for example) but overall it was a clean, clear story that made sense. When Lee Babiss wrote Ginsberg’s obituary in 2003⁴ he included this among Ginsberg’s particularly important acheivements:

They also began to investigate the role in pathology of the adenoviral early gene 3 region, and determined that the proteins encoded by the E3 transcripts influenced the host inflammatory response. This observation led the way to the creation of adenoviral gene delivery vectors that could persist in the host cell for long periods of time, thus promoting prolonged transgene expression.

The problem is that it’s likely not true.

Counterevidence
The E3gp19k mutants were naturally-occurring mutants with actual deletions in the E3 region. There’s no obvious reason why this should present a problem, but an obscure paper in 1994 showed that in fact, it is a problem:

Berencsi, K., Uri, A., Valyi-Nagy , T., Valyi-Nagy, I., Meignier, B., Peretz, F.V., Rando, R.F., Plotkin, S.A., Gönczöl, E. (1994). Early region 3-replacement adenovirus recombinants are less pathogenic in cotton rats and mice than early region 3-deleted viruses. Laboratory Investigations, 71(3), 350-358.

I admit that I haven’t read the whole paper yet. It’s only available on paper (how quaint!) and my request to the library for a copy hasn’t been answered yet. Still, the abstract is very clear. The authors compared the deletion mutant with a replacement mutant — still eliminating E3gp19k, but replacing it with an unrelated gene that restores the genome size to normal (actually greater than normal). The replacement mutant doesn’t show the pathology that the deletion mutant does.

An Ad5 recombinant, Ad-human cytomegalovirus glycoprotein B (Ad-HCMV.gB), in which the E3 region is replaced by the full-length gB gene of HCMV and with a genome size exceeding that of Wt-Ad, induced mild histopathologic responses in cotton rat and mouse lungs, comparable with those of Wt-Ad, but less severe than those of Ad5-delta E3. Analysis indicated that neither class I major histocompatibility complex expression on the cell surface nor differential expression of the protective E3-14.7 kilodalton protein underlies the pathologic differences observed in cells infected with Ad5-delta E3 or the Ad-HCMV.gB recombinant. … Pathogenicity and replication of the recombinant viruses inversely correlate with the genomic size.

(My emphasis.) The lung inflammation is a genome size effect, not an E3gp19k effect.

What’s more, this has been reproduced. In 2001, a second group found exactly the same thing using adenovirus type 4 instead of type 5. ⁵ They didn’t even know about the Berencsi paper, and didn’t make a connection to genome size (they floundered about trying to explain the effect as a function of the inserted genes), but the actual observation was essentially exactly the same: Pathogenicity is related to reduced genome size, but not to loss of E3gp19k.

As found previously for Ad5, deletion of Ad4 E3 genes resulted in increased lung pathology. Surprisingly, insertion of HIV genes into this region significantly restored protection attributed to E3 gene products, diminishing overall pathologic effects to Ad4WT levels (P<= 0.0001).

Ginsberg’s original paper has been cited over 200 times. Berencsi’s has been cited just 13 times, mainly for technical aspects; as far as I can tell none of the citations actually note the critical observation. Patterson’s paper? Only 4 citations (and as I say, they themselves didn’t cite Berencsi either). Fields Virology, the authoritative source, mentions Berencsi et al in passing but doesn’t describe or comment on the actual finding, let alone its significance, and doesn’t mention Patterson et al at all. Fields offers the party line, Ginsberg’s interpretation, on pulmonary inflammation.

It’s possible that people in the field are aware of the observation and are discounting it for some good reason, but if so, it’s apparently an unpublished good reason. (Maybe when I see the paper I’ll decide it’s a load of dingo’s kidneys, but it’s hard to see how it could go that wrong; what’s more, the replication by Patterson et al make it much more likely that the phenomenon is real.) If it’s not E3gp19k deletion that’s causing the causing the inflammation, what is it? I have no idea. Perhaps the deletion alters regulation or expression of another gene (perhaps by altering splicing); perhaps it alters the rate of genome replication; perhaps (because this is 2008) there’s some microRNA effect. Who knows? The important thing is now I have one less piece of evidence that CTL evasion is important in vivo.

1. And apparently no one else has realized it yet[↩]
2. Pacini, D. L., Dubovi, E. J., and Clyde, W. A. J. (1984). A new animal model for human respiratory tract disease due to adenovirus. J Infect Dis 150, 92-97.[↩]
3. Ginsberg, H. S., Horswood, R. L., Chanock, R. M., and Prince, G. A. (1990). Role of early genes in pathogenesis of adenovirus pneumonia. Proc Natl Acad Sci U S A 87, 6191-6195.
   Ginsberg, H. S., Moldawer, L. L., Sehgal, P. B., Redington, M., Kilian, P. L., Chanock, R. M., and Prince, G. A. (1991). A mouse model for investigating the molecular pathogenesis of adenovirus pneumonia. Proc Natl Acad Sci U S A 88, 1651-1655.[↩]
4. Babiss, L. E. (2003). In memoriam: Harold S. Ginsberg (1917-2003). Arch Virol 148, 1655-1657.[↩]
5. Patterson, L. J., Prince, G. A., Richardson, E., Alvord, W. G., Kalyan, N., and Robert-Guroff, M. (2002). Insertion of HIV-1 genes into Ad4DeltaE3 vector abrogates increased pathogenesis in cotton rats due to E3 deletion. Virology 292, 107-113.[↩]

Cytotoxic T lymphocytes (CTL) are important destroyers of virus, recognizing MHC class I on infected cells and killing the cell before virus replication is complete. Unsurprisingly, many viruses (especially, but not only, the large and complex viruses like herpesviruses and adenoviruses) have evolved mechanisms to block MHC class I antigen processing and, therefore, T cell recognition.

What has been surprising is how ineffective these viral immune evasion mechanisms seem to be. (I’ve talked about this before in a bit more detail.) In cells in the incubator (which is where most of the experiments with the antigen presentation immune evasion molecules have been done), the effects often look pretty dramatic; but when you look at the immune response in humans infected with human cytomegalovirus¹ or herpes simplex virus² (both of which have formidable immune evasion functions in tissue culture³) there are pretty hearty and effective CTL responses, suggesting that the immune evasion doesn’t work all that well in vivo.

Most of the viruses that have had immune evasion genes identified are human viruses; that’s where the clinical interest and the money are. One problem with this is that the large DNA viruses that are likely to have these immune evasion functions tend to be pretty species-specific. Human herpesviruses and adenoviruses don’t infect mice well, if at all, and even if they do infect lab animals it’s not really clear how accurately this infection mimics the natural course in humans. There are a couple of lab animal herpesviruses that may be useful models (though even here the effects of immune evasion molecules are quite small.⁴) Still, it would be nice to understand what’s going on with immune evasion in, say, herpes simplex infection.

Herpes simplex virus actually will infect mice (the alphaherpesviruses tend to be somewhat less host-restricted than the beta or gammaherpesviruses), and while mouse infection isn’t exactly like that in humans it does seem to be similar enough to offer useful insights. Not into immune evasion, though, because the immune evasion gene of herpes simplex (called “ICP47″) works very poorly in mice.⁵ Recently, though, Chris Wilson’s group took an ingenious approach to looking at the question, and a theme may be starting to emerge.

Wilson reasoned that if ICP47 doesn’t work in mice,⁶ why not replace it with a functionally-similar gene that does? They made recombinant herpes simplex viruses that have immune evasion genes from mouse or human cytomegalovirus that block mouse MHC class I fairly well. These viruses are more neurovirulent in mice than the control viruses⁷ and this was because the CTL didn’t work as well in the recombinants. ^8,9

Their latest paper¹⁰ shows something new and exciting with the recombinant virus: It reactivates better from neurons.

Trigeminal ganglion

Remember that HSV establishes a latent infection in neurons and, in humans, periodically reactivates and may shed and infect new hosts this way. HSV latency is still a mysterious and little-understood field in spite of huge amounts of work over the years, but a host of recent findings have started to explain at least bits and pieces. It used to be felt that latent HSV didn’t express any proteins, and so should be invisible to the immune system. As I observed here, it’s now known that the immune system can detect HSV in neurons. That offers a mechanism for reactivation to be coupled with immune suppression. The Orr et al paper here offers more support for this concept, and suggests that one of the (most important?) functions for HSV immune evasion is to tip the scales a little further toward the virus being able to escape recognition in the neuron, and therefore being more easily able to reactivate and infect new hosts — something this ubiquitous virus does with extraordinary efficiency. Orr et al call this an “actively testing the waters” model, in which HSV constantly sticks its toes out into the stream of the immune response to see if it’s safe to reactivate.

What especially interesting about this is that we now have (that I know of) three models of MHC class I immune evasion in more or less authentic infections, and each case the immune evasion seems to be designed to support latency, reactivation, and transmission. Here we have HSV using immune evasion in latency and persistence. Ann Hill’s group has recently shown that mouse cytomegalovirus MHC class I immune evasion proteins help the virus persist in the salivary glands,¹¹ so that (as with the HSV story) it is more able to infect new hosts. And in the third model I know of, mouse herpesvirus 68, the MHC class I immune evasion genes are important for, you guessed it, latency and persistence.¹²

Three data points aren’t very many, but it does suggest that this may be a common feature of the herpesviruses. Perhaps in acute infection the virus doesn’t really “want” to block immune responses all the effectively, because their lifestyle involves only a brief burst of acute infection followed by a long-term latency; and it’s that latency that allows infection of new hosts. The viruses “let” themselves be driven into apparent submission, but then use their immune evasion functions to mount intermittent stealth campaigns to recruit new hosts.

If this is true for herpesviruses (and of course it’s just a hypothesis) is it also true for other viruses that have MHC class I immune evasion functions? Maybe for some (I wonder about the adenoviruses in particular, since they’re also very capable of long-term persistence in spite of immune responses) but I doubt it’s universal — HIV, for example, doesn’t obviously fit into the same lifestyle pattern as the herpesviruses, and yet if has a protein (nef) that targets MHC class I. Still, if offers a framework for thinking about the problem, which is very valuable.

1. Manley, T. J., Luy, L., Jones, T., Boeckh, M., Mutimer, H., and Riddell, S. R. (2004). Immune evasion proteins of human cytomegalovirus do not prevent a diverse CD8+ cytotoxic T-cell response in natural infection. Blood 104, 1075-1082.[↩]
2. Posavad, C. M., Koelle, D. M., and Corey, L. (1996). High frequency of CD8+ cytotoxic T-lymphocyte precursors specific for herpes simplex viruses in persons with genital herpes. J. Virol. 70, 8165-8168.[↩]
3. York, I. A., Roop, C., Andrews, D. W., Riddell, S. R., Graham, F. L., and Johnson, D. C. (1994). A cytosolic herpes simplex virus protein inhibits antigen presentation to CD8+ T lymphocytes. Cell 77, 525-535.
   and
   Jones, T. R., Hanson, L. K., Sun, L., Slater, J. S., Stenberg, R. M., and Campbell, A. E. (1995). Multiple independent loci within the human cytomegalovirus unique short region down-regulate expression of major histocompatibility complex class I heavy chains. J. Virol. 69, 4830-4841.[↩]
4. For example, Munks, M. W., Pinto, A. K., Doom, C. M., and Hill, A. B. (2007). Viral interference with antigen presentation does not alter acute or chronic CD8 T cell immunodominance in murine cytomegalovirus infection. J Immunol 178, 7235-7241.[↩]
5. Jugovic, P., Hill, A. M., Tomazin, R., Ploegh, H., and Johnson, D. C. (1998). Inhibition of major histocompatibility complex class I antigen presentation in pig and primate cells by herpes simplex virus type 1 and 2 ICP47. Journal of Virology 72, 5076-5084.[↩]
6. To be strictly accurate, ICP47 does work in mice, just not very well — 100 times less than in humans, maybe, though what the unit of comparison is or should be isn’t clear[↩]
7. I first wrote “than wild-type virus”, but I don’t think we know that — the process of recombination made the virus less virulent and the comparisons were done to this reduced-virulence control virus; I don’t see a direct comparison to wild-type virus.[↩]
8. Orr, M. T., Edelmann, K. H., Vieira, J., Corey, L., Raulet, D. H., and Wilson, C. B. (2005). Inhibition of MHC class I is a virulence factor in herpes simplex virus infection of mice. PLoS Pathog 1, e7.[↩]
9. This actually parallels and supports previous work showing that ICP47 does in fact have a small effect on neurovirulence in mice.[↩]
10. ORR, M., MATHIS, M., LAGUNOFF, M., SACKS, J., WILSON, C. (2007). CD8 T Cell Control of HSV Reactivation from Latency Is Abrogated by Viral Inhibition of MHC Class I. Cell Host & Microbe, 2(3), 172-180. DOI: 10.1016/j.chom.2007.06.013 [↩]
11. Lu, X., Pinto, A. K., Kelly, A. M., Cho, K. S., and Hill, A. B. (2006). Murine cytomegalovirus interference with antigen presentation contributes to the inability of CD8 T cells to control virus in the salivary gland. J Virol 80, 4200-4202.[↩]
12. Bennett, N. J., May, J. S., and Stevenson, P. G. (2005). Gamma-herpesvirus latency requires T cell evasion during episome maintenance. PLoS Biol 3, e120.[↩]