Mystery Rays from Outer Space

I’m not so much an antibody guy, but of course I’ve heard about catalytic antibodies. Catalytic antibodies bind, with the very high affinity that’s typical of many antibodies, to transition state molecules, stabilizing the transition state and facilitating the chemical reaction. They’ve been around for quite a while (I think the first, or at least first widely-announced, catalytic antibody¹ was described in the mid-1980s) and a fair number of them have been created.

But as far as I know, catalytic antibodies remain a curiosity — a fascinating curiosity, but one without a lot of practical application. Although they can be custom-made and can have great specificity, as enzymes they tend to be really crappy (as you might expect), acting thousands or millions of times slower than “genuine” enzymes. Again, I’m no expert, but it seems that in spite of decades of promise, there hasn’t been much payoff; which is a shame, because the concept is so cool they deserve to make it big.²

Recently, though, there was a paper³ offering a catalytic antibody that actually was therapeutic in a real live animal model. Is this the one where promise actually follows through to reality?

The antibody was raised against a virulence factor, urease, of Helicobacter pylori, the ulcer-causing bacterium. (Urease helps neutralize the stomach acid, so it helps H. pylori colonize stomachs.) The antibody degraded urease reasonably well; and more remarkably, the light chain alone of the antibody was also able to degrade H. pylori urease. I don’t know how common this is for catalytic antibodies,⁴ but it strikes as potentially useful, since the light chain can be more readily synthesized and is probably more stable on its own.

The interesting part was that they treated mice with this — I think the first time catalytic antibodies have been used in vivo. They infected the mice with H. pylori, and then treated them with either the catalytic light chain in buffer, or with buffer alone (they should really have used a control light chain in buffer, though — in general this paper doesn’t strike me as terribly well-controlled when is comes to the animal work, but part of that may be the poor English throughout). The treated mice had about a third as many bacteria in their stomachs as did control mice, suggesting that the antibody actually did some good. Presumably it degraded urease in vivo as it does in vitro, and by inceasing stomach acidity helped reduce the bacterial survival.

This is still far, far from any kind of useful treatment, I think, but it’s a step forward.

1. Pollack SJ, Jacobs JW, Schultz PG (1986) Selective chemical catalysis by an antibody. Science 234:1570-1573.[↩]
2. If I’m wrong, by the way, and catalytic antibodies have achieved a toehold somewhere in medicine or industry, please let me know. As I say, I don’t follow this field all that closely.[↩]
3. Hifumi, E., Morihara, F., Hatiuchi, K., Okuda, T., Nishizono, A., Uda, T. (2007). Catalytic Features and Eradication Ability of Antibody Light-chain UA15-L against Helicobacter pylori. Journal of Biological Chemistry, 283(2), 899-907. DOI: 10.1074/jbc.M705674200[↩]
4. It’s not unique, because the authors say they had the same thing for a previous catalytic antibody they made[↩]

One of the genes I’m interested in is an aminopeptidase called “ERAP1″¹ that’s involved in antigen presentation.² ERAP1 is a member of a family of closely-related genes, roughly 50% identity, that in humans and many other mammals are adjacent on a chromosome. That is, on human chromosome 5 (figure to the right), there are three genes side by side: ERAP1; ERAP2; and LNPEP, all of which are clearly the result of gene-duplication events at some point. Several other mammals have the same arrangement. The neighbouring (non-ERAP-related) genes are the same, also, for that matter.

In mice and rats, however, there’s a different arrangement. ERAP1 is on chromosome 13, LNPEP is on chromosome 17, and there’s no ERAP2.

So it seemed that the most likely scenario was the in the rodent lineage, there was a chromosome break right between ERAP1 and LNPEP, right in the middle of ERAP2, that destroyed ERAP2. However, it was also possible (though I thought less likely) that there was a fusion — that having ERAP1 and LNPEP on different chromosomes was the basal situation, but that the chromosomes merged in the non-rodent lineage, with a gene duplication arising from the chromosome fusion. Or something like that.

Fish have members of the ERAP family, but I wasn’t confident in assigning them to ERAP1, ERAP2, or LNPEP — the mammalian genes are too similar for me to be confident in my feeble bioinformatic understanding.

But the platypus genome that just come out gives a lot of support to the idea that rodents split their chromosome from the basal patterns. To the left (click for a larger version) is the appropriate platypus chromosome. ERAP1 (here called “similar to type I tumor necrossis factor receptor shedding aminopeptidase regulator”) and LNPEP (here called “similar to leucyl/cystinyl aminopeptidase”) are on the same chromosome, and in between them is “LOC10081647″ — something the annotation called a pseudogene, which it may well be, but it’s ERAP2. I haven’t had time to do any careful alignments, but it aligns more closely with mammalian ERAP2 than anything else.

So almost certainly mice and rats lost ERAP2, though it may have been a pseudogene at that point, and other species either kept a functional ERAP2, or somewhere along the line turned a pseudogene into a functional enzyme.

This is not a big deal and likely won’t lead anywhere, but it’s an answer to a piece of trivia that’s been bothering me a little on and off for about 5 years. So yay for the platypus genome.

1. by us, anyway: York IA, Chang SC, Saric T, Keys JA, Favreau JM, Goldberg AL, Rock KL (2002) The ER aminopeptidase ERAP1 enhances or limits antigen presentation by trimming epitopes to 8-9 residues. Nat Immunol 3:1177–184. [↩]
2. For example, York IA, Brehm MA, Zendzian S, Towne CF, Rock KL (2006) Endoplasmic reticulum aminopeptidase 1 (ERAP1) trims MHC class I-presented peptides in vivo and plays an important role in immunodominance. Proc Natl Acad Sci U S A 103:9202–9207.[↩]

I’ve talked several times about Charlie Janeway’s “dirty little secrets“, and the insights into fundamental immunity that arose from the concept. I’ve also mentioned a couple of potential clinical advances arising from it. Here’s another one, that I find particularly elegant for its use of the weak to conquer the powerful. ¹

As a very quick reminder: Janeway’s insight² was that an immune response wouldn’t start unless there were signals present, indicating that a hazardous situation was at hand. Janeway proposed that the immune system would be on the alert for molecular patterns that are generic to many pathogens. Without such patterns the immune system would ignore “foreign” antigen; when pathogen-associated molecular patterns (”PAMPs”) appear, the immune system kicks on and starts looking for trouble. (By the way, sorry about all the acronyms in this. I usually try to avoid using too many, but it’s unavoidable this time. There’s a glossary in the footnote here if you need it.)³

Janeway, and subsequently many others, went on to identify some of the PAMP receptors; first the toll-like receptors (TLRs) and then several other types. There are quite a few — maybe a dozen TLRs, maybe a couple dozen other types, in mice or humans. The different PAMP receptors recognize different subsets of PAMPs, and we have relatively recently reached the point where we understand enough about the receptors to make occasional predictions: Researchers can analyze a virus, say, and say with some confidence that a certain PAMP receptor is likely to recognize it.

Immune recognition of mousepox virus
Hubertus Hochrein’s group is interested in smallpox, the archetypal poxvirus, and they’re using ectromelia (mousepox) as their model for smallpox. Poxviruses are large DNA viruses that are remarkably versatile in their dealings with the immune system; as a group, and as individual viruses, they have evolved molecules that evade multiple components of the immune system. One of those components is the TLR system, apparently, because at least some poxviruses encode molecules that block TLR signalling. ⁴

There’s an interesting general question, by the way, about how to interpret immune evasion molecules in viruses. If we find that vaccinia virus encodes blockers of TLR signaling, do we argue that TLRs must be important in protecting against vaccinia virus? Or do we instead say that TLRs must not be important, because the virus has defenses against them? In this case, at any rate, Hochrein’s group guessed that TLRs are important, and further guessed that TLR9 might be important.

TLR9 recognizes DNA, both viral and bacterial, but until now there haven’t been any instances of virus recognition that’s strictly dependent on TLR9. Ectromelia, however, turned out to be the first; immune activation by ectromelia is almost entirely dependent on TLR9 signaling, and mice lacking TLR9 were highly sensitive to ectromelia infection:

The in vivo relevance of this TLR9-only dependence for ECTV⁵ recognition was clearly illustrated by our in vivo studies that revealed that the lack of TLR9 rendered mice more than 100-fold more susceptible to infection with ECTV. … We calculated an LD₅₀⁶ of 19 TCID₅₀⁷ for the TLR9-deficient mice and an LD₅₀ of about 2,120 TCID₅₀ for the WT mice.

Cells (actin cytoskeleton in green) infected with vaccinia virus (red)

Broader recognition of a weakened poxvirus
Does TLR9, and only TLR9, recognize poxviruses in general? Ectromelia is a highly virulent virus even as poxviruses go. There are plenty of more benign viruses, such as vaccinia virus; and even within vaccinia viruses there is a wide range of virulence. Probably the least virulent vaccinia virus is a semi-artificial version of it called “Modified vaccinia Ankara” (MVA). ⁸ MVA has lost about 13% of its genome compared to its more virulent ancestor, and many of its remaining genes are damaged as well.⁹

Like ectromelia, TLR9 drove an immune response to MVA. Unlike ectromelia, that isn’t the whole story; even without TLR9, the immune system recognizes MVA.

This is almost certainly an immune evasion function that has been lost in MVA. That is, both wild-type vaccinia virus and ectromelia virus seem to have a gene (or genes) that blocks recognition by PAMP receptors other than TLR9, whereas the massively defective MVA has lost this gene and is recognized by both TLR9 and this other, unknown, receptor.

Overriding blindness
So if immune activation by ectromelia is partially blocked by its immune evasion function, would we reduce its virulence by artificially activating the immune system after ectromelia infection? Ideally, of course, we’d want to only activate the components that are involved in protecting against poxviruses. Like, for example, the aspects that the poxvirus MVA activates.

You see where this is going. Can MVA act almost like an adjuvant, turning on the immune components that ectromelia virus has blinded? And the answer is yes. If you infect mice with a lethal dose of ectromelia, and then superinfect them with MVA, they survive:

MVA given at the same time or immediately after challenge with a high lethal dose of ECTV of 1 × 10⁵ TCID₅₀ completely protected WT mice against death, whereas all control mice died with the 10-fold-lower dose of 1 × 10⁴ TCID₅₀.

You wouldn’t normally think that two viruses would be better than one; and you wouldn’t normally think that the dainty little MVA could override its brutally virulent cousin’s lethality. But at least in mice, it seems that therapeutic infection worked.

1. Samuelsson, C., Hausmann, J., Lauterbach, H., Schmidt, M., Akira, S., Wagner, H., Chaplin, P., Suter, M., O’Keeffe, M., Hochrein, H. (2008). Survival of lethal poxvirus infection in mice depends on TLR9, and therapeutic vaccination provides protection. Journal of Clinical Investigation, 118(5), 1776-1784. DOI: 10.1172/JCI33940[↩]
2. And Polly Matzinger’s[↩]
3. PAMP: Pathogen-associated molecular pattern;
   TLR: toll-like receptor;
   ECTV: ectromelia virus;
   LD₅₀: Dose of virus that kills half the recipients;
   TCID₅₀: 50% tissue-culture infectious dose - more or less, the number of infectious particles of virus;
   MVA: Modified vaccinia Ankara[↩]
4. Bowie A, Kiss-Toth E, Symons JA, Smith GL, Dower SK, O’Neill LA (2000) A46R and A52R from vaccinia virus are antagonists of host IL-1 and toll-like receptor signaling. Proc Natl Acad Sci U S A 97:10162-10167.[↩]
5. ectromelia virus[↩]
6. LD₅₀: Dose of virus that kills half the recipients.[↩]
7. TCID₅₀: 50% tissue-culture infectious dose - more or less, the number of infectious particles of virus[↩]
8. MVA was produced by repeatedly passing a wild vaccinia virus (Ankara strain) through chicken cells more then 570 times. In the process of becoming chicken-adapted, it lost its mammalian adaptations and barely replicates in mammalian cells. Since it’s so enfeebled, there’s interest in using it as a vaccine, since the standard smallpox vaccine is quite dangerous as vaccines go.[↩]
9. Meisinger-Henschel C, Schmidt M, Lukassen S, Linke B, Krause L, Konietzny S, Goesmann A, Howley P, Chaplin P, Suter M et al. (2007) Genomic sequence of chorioallantois vaccinia virus Ankara, the ancestor of modified vaccinia virus Ankara. J Gen Virol 88:3249-3259.[↩]

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.[↩]

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.[↩]

We know that HIV can be controlled by an appropriate immune response. Cytotoxic T lymphocytes (CTL) are capable of very effectively suppressing HIV; in fact, in a standard HIV infection, the virus typically spends most of its early phase being controlled by a T cell response. In most people, unfortunately, the control is temporary; since HIV replication is sloppy, the virus throws off mutants at regular intervals, and eventually one of the mutants will be invisible to the dominant CTL response. That mutant replicates rapidly (probably damaging the immune response as it does so) until a new CTL response brings that virus under control, only for other variants to arise again.

Some people are apparently able to hold the virus under control for very long periods — the long-term non-progressor HIV patients. Some of these people seem to have T cell responses against part of the virus that has very precise sequence requirements; if the virus mutates away from CTL recognition, the virus is crippled and can’t replicate effectively. Other people seem to have a broad T cell response, one that recognizes several parts of the virus at once. The odds of successfully mutating all of the targeted areas simultaneously are exponentially lower than of mutating a single region.

Obviously, either of these are states that vaccine designers want as outcomes. That’s not all that easy. People are variable, and there don’t seem to be general rules that you can use to force an immune response to the target of one’s choice. ¹ Wouldn’t it be nice if there was a way of bypassing the whole messy immunization step, and just moving straight on to the desired finale of CTL specific for the target of one’s choice?

A paper in the March ‘08 issue of Journal of Virology² does just that.

When you induce T cell-mediated immunity, whether through a vaccine or a real infection, what you’re actually doing is expanding a pool of T cells whose receptor recognizes your special antigen. There are a huge number of potential T cell receptors (TcRs); under normal conditions, any particular antigenic target might have only 20 or 100 T cells that can recognize it, scattered among the millions of T cells with irrelevant specificities. Once a T cell finds its antigen, though,³ that T cell clone divided and expands enormously, as much as 100,000 times. The next time that antigen rides through town, it finds hundreds of sheriffs awaiting it, not just one or two.

If the TcR is all you need for specific recognition, can you bypass the whole annoying specific recognition and expansion step? Why not take the TcR from a previous clone, that you already know is useful (perhaps one from another individual altogether) and swap it into generic, non-specific T cells? In fact, that’s been done in a number of cases, and it actually seems to work.⁴

Joseph et al. tried this with a TcR specific for a HIV antigen. They swapped this known TcR into ordinary generic T cells from a normal blood donor, and turned those boring old plain T cells into CTL that specifically killed HIV-infected cells.

OK, their system is very artificial, involving transformed target lines and a Rube Goldbergesque mouse system to test “in vivo activity”, so it’s not really possible to draw any conclusions about clinical potential. In an actual infection, you’d presumably want to do this with multiple TcRs simultaneously, to target many HIV antigens at once and reduce the risk of immune escape (otherwise, just putting in one chimeric TcR is not different from getting a strong CTL response to HIV — which we know is not sufficient in the long run). I don’t think we know what would happen in that situation; would there be competition between the different TcRs to the point that most would be outcompeted and swamped, ending up with a de facto single target after all? ⁵

Another question I have is whether the original TcRs might cause mischief — if the T cell has two TcRs, stimulation through one might lead to reactivity with the other, and if the other, original, TcR happens to react with a self antigen you might get the mother of all autoimmune diseases. So my guess is that this is mostly a cute idea that will never go anywhere (for HIV; I think it has much more potential in tumor treatment).

Still, it really is a neat concept, and I hope some of my questions get addressed.

1. There are some approaches that can do this, but they also have drawbacks.[↩]
2. Joseph, A., Zheng, J.H., Follenzi, A., DiLorenzo, T., Sango, K., Hyman, J., Chen, K., Piechocka-Trocha, A., Brander, C., Hooijberg, E., Vignali, D.A., Walker, B.D., Goldstein, H. (2008). Lentiviral Vectors Encoding Human Immunodeficiency Virus Type 1 (HIV-1)-Specific T-Cell Receptor Genes Efficiently Convert Peripheral Blood CD8 T Lymphocytes into Cytotoxic T Lymphocytes with Potent In Vitro and In Vivo HIV-1-Specific Inhibitory Activity. Journal of Virology, 82(6), 3078-3089. DOI: 10.1128/JVI.01812-07[↩]
3. assuming appropriate conditions for activation and so forth[↩]
4. E.g. for tumors; Morgan, R. A., Dudley, M. E., Wunderlich, J. R., Hughes, M. S., Yang, J. C., Sherry, R. M., Royal, R. E., Topalian, S. L., Kammula, U. S., Restifo, N. P., Zheng, Z., Nahvi, A., de Vries, C. R., Rogers-Freezer, L. J., Mavroukakis, S. A., and Rosenberg, S. A. (2006). Cancer regression in patients after transfer of genetically engineered lymphocytes. Science 314, 126-129.[↩]
5. Some models for immunodominance predict this, in fact[↩]

What with visiting speakers and new faculty recruitment, I’ve been out late every night this week; what with committee meetings¹ and trying to squeeze in experiments, I’ve been up early every morning; and what with teaching starting up again, seminars from visiting speakers and recruitees, and faculty meetings, I’ve had little time for other stuff. So this is going to be a short post. ²

A while ago I talked about lamprey immune systems. The key points are that lampreys …

• have an immune system
• that works pretty well
• and in concept looks a lot like our immune system, with lymphocytes and specific receptors
• but the receptors are utterly unlike our T cell receptors and antibodies
• using instead of the immunoglobulin domain structure, a leucine-rich repeat (LRR) structure;
• and, instead of using RAG-based recombination, uses a gene conversion system to generate diversity.

There’s a temptation, even for those who intellectually know better, to assume that “primitive” animals have “worse” systems; so because lampreys are more like the common ancestor of vertebrates, their immune system must be “worse”. (Hagfish and lampreys, which may have diverged some some 500 million years ago — see the figure to the right;³ click for a larger version — have very similar immune systems, so this system must be at least that old.) In some ways the mammalian immune response does seem to have some advantages — faster memory response, for example. Still, lamprey immune systems have served them well for 500 million years, which is more than we can say about ours; and in some other ways lampreys do better than we do. They have if anything a greater diversity to their receptors, for example, potentially generating more than 10¹⁴ different receptors — compare to our roughly 10⁸ T cell receptors.

Max Cooper, who has done much of the work on lamprey immunity,⁴ has just published a paper showing off some other unusual properties of lamprey immune receptors. ⁵ Since there’s no system for making lamprey monoclonal antibodies that’s analogous to the mouse monoclonal antibody systems, he used a molecular cloning approach to express monoclonal variable lymphocyte receptor (VLR) -B cDNAs from immunized lampreys.

What did they get?

They got soluble “antibodies”, capable of the highly specific recognition that’s seen in conventional monoclonal antibodies. The VLR-B antibodies are extraordinarily stable, maintaining binding at pH 1.5 and maintaining structure at pH 11, as well as after incubation at 56 ^oC for a couple days or at room temperature for weeks. ⁶ Although the individual LRR subunits have relatively low binding affinity, they are secreted as multimers of eight to ten subunits (see the diagram to the left), and as a result the VLR-B binding ability can be at least as good as mouse monoclonals: “Equal concentrations of VLR4 and EA2-1, starting at 0.5 mg/ml, were serially diluted in 10-fold increments and scored for the degree of spore agglutination. Spore agglutination by VLR4 was detected at a concentration 1,000-fold more dilute (5 pg/ml) than the mouse monoclonal antibody (5 ng/ml).”

Finally, as opposed to mouse monoclonals, these are single proteins; conventional mouse monoclonals have two components, a heavy and a light chain. That makes VLR-B easier to work with in some ways: “The single peptide composition of VLR-B antibodies makes them more amenable to molecular engineering, including manipulation of the antigen binding site by mutagenesis and fusions to the coding sequences of other peptides, such as enzymes, toxins, and epitope tags to extend their functional capabilities.” ⁷

These things clearly have potential to be useful in all kinds of things — a nice example of basic research giving rise to clinically and commercially useful tools.

1. Proof, if proof were needed, that deans are evil: 7:30 AM meeting with the dean[↩]
2. Also, I just realized I really, really need to get some flowers for my wife today, don’t I.[↩]
3. From: Modern look for ancient lamprey. Philippe Janvier. Nature 443, 921-924(26 October 2006 [↩]
4. It was his talk at the Autumn Immunology Conference in Chicago a couple of years ago that made me realize how fascinating the subject is[↩]
5. Herrin, B.R., Alder, M.N., Roux, K.H., Sina, C., Ehrhardt, G.R., Boydston, J.A., Turnbough, C.L., Cooper, M.D. (2008). Structure and specificity of lamprey monoclonal antibodies. Proceedings of the National Academy of Sciences, 105(6), 2040-2045. DOI: 10.1073/pnas.0711619105[↩]
6. Conventional antibodies are pretty stable, but not up to this level.[↩]
7. I think it’s camels that make single-chain antibodies, and there’s been interest in developing monoclonal systems based on camel antibodies for the same reason.[↩]

TcR interacting with artificial membrane1

Earlier this week I talked about the phenomenon of viruses that downregulate MHC class II. The “purpose” of this blockade is kind of unclear to me, because the immunity driven by MHC class II is not focused on the cell it’s attached to, but rather spills out broadly over a wide area; it seems that a virus would have to very rapidly infect a very large number of MHC class II-expresing cells to have much effect on the anti-viral immune system.

One possible explanation is that the downregulation may be only peripherally related to MHC class II-based immunity. Instead, the virus could be simply going about its cell-biological business and either targeting MHC class II as an accidental side-effect, or because of some function of MHC class II that isn’t directly related to immunity. Here’s a parallel case that may help think about the problem.

The paper is
Sullivan, B.M., Coscoy, L. (2007). Downregulation of the T-Cell Receptor Complex and Impairment of T-Cell Activation by Human Herpesvirus 6 U24 Protein. Journal of Virology, 82(2), 602-608. DOI: 10.1128/JVI.01571-07

The T-cell receptor (TcR) is what recognizes MHC class I or II. Human herpesvirus 6 (HHV6) infects T helper (CD4) cells and (depending on viral strain) also does a number of things to modulate the immune system.² Sullivan and Coscoy show here that the virus also down-regulates the TcR on infected T cells. (It’s altogether a more solid paper than the last one I mentioned, with nice experiments that directly show what’s happening to the TcR: “HHV-6 U24 protein inhibits CD3 recycling to the cell surface and, as a consequence, downregulates CD3 cell surface expression and prevents T-cell activation.“)

U24 blocks CD3ε access to Rab11 recycling endosomes.3

At first glance this raises exactly the same question as does Vpu’s effect on MHC class II. How does reducing TcR on infected cells benefit the virus? The infected T cell will be less able to recognize its target, but what are the odds that its target is HHV6? Pretty minimal; there are (at least) tens of billions of different TcRs and only a handful of them recognize any particular antigen. The virus might be causing generalized immune suppression if it infects a large fraction of the T cells, but that’s not a particular benefit for the virus. If HHV6 specifically infected cells with TcRs that are specific for the virus then this would be a targeted immune evasion technique, but as far as I know there’s no evidence for this, nor is there an obvious mechanism by which HHV6 could target antigen-specific CD4 T cells.

There is, however, a nice explanation for TcR downregulation that doesn’t involve direct effects on T cell recognition. HHV6 (like all herpesviruses) has two choices when it infects a cell. It can either enter lytic replication — replicating the genome, producing more viruses, and eventually destroying the infected cell — or enter latency — a long-term, perhaps life-long, infection with minimal protein expression and little if any effect on the infected cell. As with any virus, the more prepared a cell is to replicate, the easier it is for a virus to replicate it’s own genome. T cells that receive a signal through their TcR become activated⁴ and divide very rapidly. In this environment, it’s very easy for HHV6 to replicate — that is, to enter lytic replication and kill the cell, releasing more viruses into the system.

Human herpesvirus 6

Probably HHV6 downregulates the TcR “because” it prevents its host from becoming activated by whatever its random antigen is. That prevents the virus from entering lytic replication and allows it to enter a persistent state, where it can hang about and await the best opportunity to infect a new person.

I know of one other viral protein that downregulates the TcR — the herpesvirus saimiri Tip protein ⁵ — and there seems to be controversy⁶ over whether this protein activates T cell signalling (potentially driving the virus into lytic replication) or blocks it (preventing lytic replication and facilitating persistence). ⁷ The original paper describing the TcR downregulation found that Tip blocked signaling, and proposed the same explanation as Sullivan and Coscoy:

… these associations ultimately block lymphocyte receptor signal transduction. … these interactions likely play an important role in the establishment and maintenance of HVS persistent infection by protecting infected cells from surveillance by the immune system. In fact, animals infected with recombinant HVSΔTip have been shown to have higher levels of cell-associated infectious virus titer compared to other recombinant HVS.

So in this case the downregulation of the TcR (a quintessentially immune molecule) apparently isn’t directly related to immune evasion, but is a way of switching between the lytic and the persistent lifestyles. (It’s also a reminder of the fairly obvious point that we shouldn’t think of viruses as blind replicators, desiring nothing more than maximal replication. At least some viruses have a range of lifestyle options, and can switch between them quite comfortably.)

I still don’t see a direct analogy to the MHC class II downregulation imposed by the HIV Vpu protein. but it’s an example of why we shouldn’t get too focused on single causes and single functions. Life is more complicated than that.

1. By Raghuveer Parthasarath, then in the Groves lab[↩]
2. Lusso, P., 2006. HHV-6 and the immune system: mechanisms of immunomodulation and viral escape. Journal of Clinical Virology, 37(Supplement 1), p.S4-S10. doi:10.1016/S1386-6532(06)70004-X [↩]
3. Sullivan & Coscoy, Fig 4[↩]
4. I am simplifying immensely![↩]
5. Park, J. et al., 2002. Herpesviral Protein Targets a Cellular WD Repeat Endosomal Protein to Downregulate T Lymphocyte Receptor Expression. Immunity, 17(2), p.221-233. doi:10.1016/S1074-7613(02)00368-0 [↩]
6. Brinkmann, M.M. & Schulz, T.F., 2006. Regulation of intracellular signalling by the terminal membrane proteins of members of the Gammaherpesvirinae. J Gen Virol, 87(5), p.1047-1074. DOI 10.1099/vir.0.81598-0[↩]
7. I’m more convinced by the argument for blocking signaling, but only because of the very bad reason that I know the people involved. I haven’t looked at the papers pro and con very carefully.[↩]

Oncolytic VSV (gold) infecting lung tumors1

Oncolytic viruses are a concept I’d like to be more excited by than I am.